Compositions and methods for inhibiting expression of the alas1 gene

ABSTRACT

The invention relates to double-stranded ribonucleic acid (dsRNA) compositions targeting the ALAS1 gene, and methods of using such dsRNA compositions to alter (e.g., inhibit) expression of ALAS1.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 16/142,953, filed Sep. 26, 2018, which is a divisional of U.S. application Ser. No. 14/814,911, filed Jul. 31, 2015, now U.S. Pat. No. 10,125,364, issued Nov. 13, 2018, which is a divisional of U.S. application Ser. No. 13/835,613, filed Mar. 15, 2013, now U.S. Pat. No. 9,133,461, issued Sep. 15, 2015, which claims priority to U.S. Provisional Application No. 61/622,288, filed Apr. 10, 2012. The entire contents of the aforesaid applications are hereby incorporated in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 14, 2016, is named A2038-719640_SL.txt and is 596,779 bytes in size.

FIELD OF THE INVENTION

The invention relates to the specific inhibition of the expression of the ALAS1 gene.

BACKGROUND OF THE INVENTION

The inherited porphyrias are a family of disorders resulting from the deficient activity of specific enzymes in the heme biosynthetic pathway, also referred to herein as the porphyrin pathway. Deficiency in the enzymes of the porphyrin pathway leads to insufficient heme production and to an accumulation of porphyrins, which are toxic to tissue in high concentrations.

Of the inherited porphyrias, acute intermittent porphyria (AIP, e.g., autosomal dominant AIP), variegate porphyria (VP, e.g., autosomal dominant VP), hereditary coproporphyria (copropophyria or HCP, e.g., autosomal dominant HCP), and 5′ aminolevulinic acid (also known as δ-aminolevulinic acid or ALA) dehydratase deficiency porphyria (ADP, e.g., autosomal recessive ADP) are classified as acute hepatic porphyrias and are manifested by acute neurological attacks that can be life threatening. The acute attacks are characterized by autonomic, peripheral, and central nervous symptoms, including severe abdominal pain, hypertension, tachycardias, constipation, motor weakness, paralysis, and seizures. If not treated properly, quadriplegia, respiratory impairment, and death may ensue. Various factors, including cytrochrome P450-inducing drugs, dieting, and hormonoal changes can precipitate acute attacks by increasing the activity of hepatic 5′-aminolevulinic acid synthase 1 (ALAS1), the first and rate-limiting enzyme of the heme biosynthetic pathway. In the acute porphyrias, e.g., AIP, VP, HCP and ADP, the respective enzyme deficiencies result in hepatic production and accumulation of one or more substances (e.g., porphyrins and/or porphyrin precursors, e.g., ALA and/or PBG) that can be neurotoxic and can result in the occurrence of acute attacks. See, e.g., Balwani, M and Desnick, R. J., Blood, 120:4496-4504, 2012.

The current therapy for the actute neurological attacks in the intravenous administration of hemin (Panhematin®, Lundbeck or Normosang®, Orphan Europe), which provides exogenous heme for the negative feedback inhibition of ALAS1, and thereby, decreases production of ALA and PBG. Hemin is used for the treatment during an acute attack and for prevention of attacks, particularly in women with the acute porphyrias who experience frequent attacks with the hormonal changes during their menstrual cycles. While patients generally respond well, its effect is slow, typically taking two to four days or longer to normalize urinary ALA and PBG concentrations towards normal levels. As the intravenous hemin is rapidly metabolized, three to four infusions are usually necessary to effectively treat or prevent an acute attack. In addition, repeated infusions may cause iron overload and phlebitis, which may compromise peripheral venous access. Although orthotrophic liver transplantation is curative, this procedure has significant morbidity and mortality and the availability of liver donors is limited. Therefore, an alternative therapeutic approach that is more effective, fast-acting, and safe is needed. It would be particularly advantageous if such treatment could be delivered by subcutaneous administration, as this would preclude the need for infusions and prolonged hospitalization.

AIP, also referred to as porphobilinogen deaminase deficiency (PBGD), or hydroxymethylbilane synthase (HMBS) deficiency, is the most common of the acute hepatic prophyrias. It is an autosomal dominant disorder caused by mutations in the HMB-synthase (HMBS) gene that result in reduced, e.g., half-normal activity of the enzyme. Previously, a mouse model of AIP that has ˜30% of wildtype HMBS activity was generated by homologous recombination Like human patients, these mice increase hepatic ALAS1 activity and accumulate large quantities of plasma and urinary ALA and PBG when administered porphyrinogenic drugs, such as phenobarbital. Thus, they serve as an excellent model to evaluate the efficacy of novel therapeutics for the acute hepatic porphyrias.

SUMMARY OF THE INVENTION

The present invention describes methods and iRNA compositions for modulating the expression of an ALAS1 gene. In certain embodiments, expression of an ALAS1 gene is reduced or inhibited using an ALAS1-specific iRNA. Such inhibition can be useful in treating disorders related to ALAS1 expression, such as porphyrias.

Accordingly, described herein are compositions and methods that effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the ALAS1 gene, such as in a cell or in a subject (e.g., in a mammal, such as a human subject). Also described are compositions and methods for treating a disorder related to expression of an ALAS1 gene, such as a porphyria, e.g., X-linked sideroblastic anemia (XLSA), ALA deyhdratase deficiency porphyria (Doss porphyria or ADP), acute intermittent porphyria (AIP), congenital erythropoietic porphyria (CEP), prophyria cutanea tarda (PCT), hereditary coproporphyria (coproporphyria, or HCP), variegate porphyria (VP), erythropoietic protoporphyria (EPP), or transient erythroporphyria of infancy. In some embodiments, the disorder is an acute hepatic porphyria, e.g., ALA deyhdratase deficiency porphyria (ADP), AIP, HCP, or VP. In certain embodiments, the disorder is ALA deyhdratase deficiency porphyria (ADP) or AIP.

In embodiments, the porphyria is a hepatic porphyria, e.g., a porphyria selected from acute intermittent porphyria (AIP) hereditary coproporphyria (HCP), variegate porphyria (VP), ALA deyhdratase deficiency porphyria (ADP), and hepatoerythropoietic porphyria. In embodiments, the porphyria is a homozygous dominant hepatic porphyria (e.g., homozygous dominant AIP, HCP, or VP) or hepatoerythropoietic porphyria, In embodiments, the porphyria is a dual porphyria.

As used herein, the term “iRNA,” “RNAi”, “iRNA agent,” or “RNAi agent” refers to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript, e.g., via an RNA-induced silencing complex (RISC) pathway. In one embodiment, an iRNA as described herein effects inhibition of ALAS1 expression in a cell or mammal.

The iRNAs included in the compositions featured herein encompass a dsRNA having an RNA strand (the antisense strand) having a region, e.g., a region that is 30 nucleotides or less, generally 19-24 nucleotides in length, that is substantially complementary to at least part of an mRNA transcript of an ALAS1 gene (e.g., a mouse or human ALAS1 gene) (also referred to herein as an “ALAS1-specific iRNA”). Alternatively, or in combination, iRNAs encompass a dsRNA having an RNA strand (the antisense strand) having a region that is 30 nucleotides or less, generally 19-24 nucleotides in length, that is substantially complementary to at least part of an mRNA transcript of an ALAS1 gene (e.g., a human variant 1 or 2 of an ALAS1 gene) (also referred to herein as a “ALAS1-specific iRNA”).

In embodiments, the iRNA (e.g., dsRNA) described herein comprises an antisense strand having a region that is substantially complementary to a region of a human ALAS1. In embodiments, the human ALAS1 has the sequence of NM_000688.4 (SEQ ID NO:1) or NM_000688.5 (SEQ ID NO:382).

In other embodiments, an iRNA encompasses a dsRNA having an RNA strand (the antisense strand) having a region that is substantially complementary to a portion of an ALAS1 mRNA according to any one of Tables 2, 3, 6, 7, 8, 9, 14, or 15. In one embodiment, the iRNA encompasses a dsRNA having an RNA strand (the antisense strand) having a region that is substantially complementary to a portion of an ALAS1 mRNA, e.g., a human ALAS1 mRNA (e.g., a human ALAS1 mRNA as provided in SEQ ID NO:1 or SEQ ID NO:382).

In one embodiment, an iRNA for inhibiting expression of an ALAS1 gene includes at least two sequences that are complementary to each other. The iRNA includes a sense strand having a first sequence and an antisense strand having a second sequence. The antisense strand includes a nucleotide sequence that is substantially complementary to at least part of an mRNA encoding an ALAS1 transcript, and the region of complementarity is 30 nucleotides or less, and at least 15 nucleotides in length. Generally, the iRNA is 19 to 24 nucleotides in length.

In some embodiments, the iRNA is 19-21 nucleotides in length. In some embodiments, the iRNA is 19-21 nucleotides in length and is in a lipid formulation, e.g. a lipid nanoparticle (LNP) formulation (e.g., an LNP11 formulation).

In some embodiments, the iRNA is 21-23 nucleotides in length. In some embodiments, the iRNA is 21-23 nucleotides in length and is in the form of a conjugate, e.g., conjugated to one or more GalNAc derivatives as described herein.

In some embodiments the iRNA is from about 15 to about 25 nucleotides in length, and in other embodiments the iRNA is from about 25 to about 30 nucleotides in length. An iRNA targeting ALAS1, upon contact with a cell expressing ALAS1, inhibits the expression of an ALAS1 gene by at least 10%, at least 20%, at least 25%, at least 30%, at least 35% or at least 40% or more, such as when assayed by a method as described herein. In one embodiment, the iRNA targeting ALAS1 is formulated in a stable nucleic acid lipid particle (SNALP).

In one embodiment, an iRNA (e.g., a dsRNA) featured herein includes a first sequence of a dsRNA that is selected from the group consisting of the sense sequences of Tables 2, 3, 6, 7, 8, 9, 14, and 15 and a second sequence that is selected from the group consisting of the corresponding antisense sequences of Tables 2, 3, 6, 7, 8, 9, 14 and 15.

In one embodiment, an iRNA (e.g., a dsRNA) featured herein has sense and/or antisense sequences selected from those of AD-58882, AD-58878, AD-58886, AD-58877, AD-59115, AD-58856, AD-59129, AD-59124, AD-58874, AD-59125, AD-59105, AD-59120, AD-59122, AD-59106, AD-59126, and AD-59107 as disclosed herein in the Examples. In embodiments, the iRNA (e.g., dsRNA) has sense and/or antisense sequences selected from those of AD-58882, AD-58878, AD-58886, AD-58877, AD-59115, AD-58856, and AD-59129.

The iRNA molecules featured herein can include naturally occurring nucleotides or can include at least one modified nucleotide, including, but not limited to a 2′-O-methyl modified nucleotide, a nucleotide having a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative. Alternatively, the modified nucleotide may be chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. Such a modified sequence can be based, e.g., on a first sequence of said iRNA selected from the group consisting of the sense sequences of Table 2, and a second sequence selected from the group consisting of the corresponding antisense sequences of Table 2.

In one embodiment, an iRNA (e.g., a dsRNA) featured herein comprises a sense strand comprising a sequence selected from the group consisting of SEQ ID NO:330, SEQ ID NO:334, SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, SEQ ID NO:356, SEQ ID NO:358, SEQ ID NO:362, SEQ ID NO:366, SEQ ID NO:376, and SEQ ID NO:380.

In one embodiment, an iRNA (e.g., a dsRNA) featured herein comprises an antisense strand comprising a sequence selected from the group consisting of SEQ ID NO:331, SEQ ID NO:335, SEQ ID NO:343, SEQ ID NO:345, SEQ ID NO:347, SEQ ID NO:357, SEQ ID NO:359, SEQ ID NO:363, SEQ ID NO:367, SEQ ID NO:377, and SEQ ID NO:381.

In one embodiment, an iRNA (e.g., a dsRNA) featured herein comprises a sense strand comprising a sequence selected from the group consisting of SEQ ID NO:140, SEQ ID NO:144, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:172, SEQ ID NO:176, SEQ ID NO:186, and SEQ ID NO:190. In one embodiment, an iRNA (e.g., a dsRNA) featured herein comprises an antisense strand comprising a sequence selected from the group consisting of SEQ ID NO:141, SEQ ID NO:145, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:173, SEQ ID NO:177, SEQ ID NO:187, and SEQ ID NO:191.

In one embodiment, an iRNA as described herein targets a wildtype ALAS1 RNA transcript variant, and in another embodiment, the iRNA targets a mutant transcript (e.g., an ALAS1 RNA carrying an allelic variant). For example, an iRNA featured in the invention can target a polymorphic variant, such as a single nucleotide polymorphism (SNP), of ALAS1. In another embodiment, the iRNA targets both a wildtype and a mutant ALAS1 transcript. In yet another embodiment, the iRNA targets a particular transcript variant of ALAS1 (e.g., human ALAS1 variant 1). In yet another embodiment, the iRNA agent targets multiple transcript variants (e.g., both variant 1 and variant 2 of human ALAS1).

In one embodiment, an iRNA featured in the invention targets a non-coding region of an ALAS1 RNA transcript, such as the 5′ or 3′ untranslated region of a transcript.

In some embodiments, an iRNA as described herein is in the form of a conjugate, e.g., a carbohydrate conjugate, which may serve as a targeting moiety and/or ligand, as described herein. In one embodiment, the conjugate is attached to the 3′ end of the sense strand of the dsRNA. In some embodiments, the conjugate is attached via a linker, e.g., via a bivalent or trivalent branched linker.

In some embodiments, the conjugate comprises one or more N-acetylgalactosamine (GalNAc) derivatives. Such a conjugate is also referred to herein as a GalNAc conjugate. In some embodiments, the conjugate targets the RNAi agent to a particular cell, e.g., a liver cell, e.g., a hepatocyte. The GalNAc derivatives can be attached via a linker, e.g., a bivalent or trivalent branched linker. In particular embodiments, the conjugate is

In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker, e.g., a linker as shown in the following schematic, wherein X is O or S

In some embodiments, X is O. In some embodiments, X is S.

In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 1 and shown below

In an aspect provided herein is a pharmaceutical composition for inhibiting the expression of an ALAS1 gene in an organism, generally a human subject. The composition typically includes one or more of the iRNAs described herein and a pharmaceutically acceptable carrier or delivery vehicle. In one embodiment, the composition is used for treating a porphyria, e.g., AIP.

In one aspect, an iRNA provided herein is a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ALAS1, wherein said dsRNA comprises a sense strand and an antisense strand 15-30 base pairs in length and the antisense strand is complementary to at least 15 contiguous nucleotides of SEQ ID NO: 1 or 382.

In a further aspect, an iRNA provided herein is a double stranded RNAi (dsRNA) comprising a sense strand complementary to an antisense strand, wherein said antisense strand comprises a region of complementarity to an ALAS1 RNA transcript, wherein each strand has about 14 to about 30 nucleotides, wherein said double stranded RNAi agent is represented by formula (III):

(III) sense: 5′ n_(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-Na-n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)- N_(a)′-n_(q)′ 5′

-   -   wherein:     -   i, j, k, and 1 are each independently 0 or 1;     -   p, p′, q, and q′ are each independently 0-6;     -   each N_(a) and N_(a)′ independently represents an         oligonucleotide sequence comprising 0-25 nucleotides which are         either modified or unmodified or combinations thereof, each         sequence comprising at least two differently modified         nucleotides;     -   each N_(b) and N_(b)′ independently represents an         oligonucleotide sequence comprising 0-10 nucleotides which are         either modified or unmodified or combinations thereof,     -   each n_(p), n_(p)′, n_(q), and n_(q)′ independently represents         an overhang nucleotide;     -   XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently         represent one motif of three identical modifications on three         consecutive nucleotides;     -   modifications on N_(b) differ from the modification on Y and         modifications on N_(b)′ differ from the modification on Y′.

In embodiments, the sense strand is conjugated to at least one ligand.

In embodiments, i is 1; j is 1; or both i and j are 1.

In embodiments, k is 1; l is 1; or both k and 1 are 1.

In embodiments, XXX is complementary to X′X′X′, YYY is complementary to Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′.

In embodiments, the Y′Y′Y′ motif occurs at the 11, 12 and 13 positions of the antisense strand from the 5′-end.

In embodiments, the Y′ is 2′-O-methyl.

In embodiments, the duplex region is 15-30 nucleotide pairs in length.

In embodiments, the duplex region is 17-23 nucleotide pairs in length.

In embodiments, the duplex region is 19-21 nucleotide pairs in length.

In embodiments, the duplex region is 21-23 nucleotide pairs in length.

In embodiments, the modifications on the nucleotides are selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl, and combinations thereof.

In embodiments, the modifications on the nucleotides are 2′-O-methyl, 2′-fluoro or both.

In embodiments, the ligand comprises a carbohydrate.

In embodiments, the ligand is attached via a linker.

In embodiments, the linker is a bivalent or trivalent branched linker.

In embodiments, the ligand is

In embodiments, the ligand and linker are as shown in Formula XXIV:

In embodiments, the ligand is attached to the 3′ end of the sense strand.

In embodiments, the dsRNA has a nucleotide sequence selected from the group of sequences provided in Tables 2 and 3. In embodiments, the dsRNA has a nucleotide sequence selected from the group of sequences provided in Tables 2, 3, 6, 7, 8 and 9. In embodiments, the dsRNA has a nucleotide sequence selected from the group of sequences provided in Tables 2, 3, 6, 7, 8, 9, 14, and 15. In embodiments, the dsRNA has a nucleotide sequence selected from the group of sequences provided in Tables 14 and 15.

In embodiments, dsRNA has a nucleotide sequence selected from the group of sequences provided in Tables 3 and 8.

In a further aspect, an iRNA provided herein is a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ALAS1, wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to an ALAS1 RNA transcript, which antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense sequences listed in any one of Tables 2, 3, 6, 7, 8, 9, 14, or 15. In some such embodiments, the sense and antisense sequences are selected from those of the duplexes AD-58882, AD-58878, AD-58886, AD-58877, AD-59115, AD-58856, AD-59129, AD-59124, AD-58874, AD-59125, AD-59105, AD-59120, AD-59122, AD-59106, AD-59126, and AD-59107 as disclosed herein in the Examples. In embodiments, the sense and antisense sequences are selected from those of the duplexes AD-58882, AD-58878, AD-58886, AD-58877, AD-59115, AD-58856, and AD-59129.

In some embodiments, the dsRNA comprises at least one modified nucleotide.

In some embodiments, at least one of the modified nucleotides is chosen from the group consisting of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.

In some embodiments, the modified nucleotide is chosen from the group consisting of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

In some embodiments, the region of complementarity is at least 17 nucleotides in length.

In some embodiments, the region of complementarity is between 19 and 21 nucleotides in length.

In some embodiments, the region of complementarity is 19 nucleotides in length.

In some embodiments, each strand is no more than 30 nucleotides in length.

In some embodiments, at least one strand comprises a 3′ overhang of at least 1 nucleotide.

In some embodiments, at least one strand comprises a 3′ overhang of at least 2 nucleotides.

In some embodiments, a dsRNA described herein further comprises a ligand.

In some embodiments, the ligand is a GalNAc ligand.

In some embodiments, the ligand targets the dsRNA to hepatocytes.

In some embodiments, the ligand is conjugated to the 3′ end of the sense strand of the dsRNA.

In some embodiments, the region of complementarity consists of an antisense sequence selected from Table 2 or Table 3. In embodiments, the region of complementarity consists of an antisense sequence selected from Tables 2, 3, 6, 7, 8, 9, 14, or 15. In some embodiments, the region of complementarity consists of an antisense sequence selected from that of AD-58882, AD-58878, AD-58886, AD-58877, AD-59115, AD-58856, AD-59129, AD-59124, AD-58874, AD-59125, AD-59105, AD-59120, AD-59122, AD-59106, AD-59126, or AD-59107 as disclosed herein in the Examples.

In some embodiments, the dsRNA comprises a sense strand consisting of a sense strand sequence selected from Table 2 or Table 3, and an antisense strand consisting of an antisense sequence selected from Table 2 or Table 3.

In some embodiments, the dsRNA comprises a sense strand consisting of a sense strand sequence selected from Tables 2, 3, 6, 7, 8, 9, 14, or 15, and an antisense strand consisting of an antisense sequence selected from Tables 2, 3, 6, 7, 8, 9, 14, or 15. In embodiments, the dsRNA comprises a pair of corresponding sense and antisense sequences selected from those of the duplexes disclosed in Tables 2, 3, 6, 7, 8, 9, 14, and 15.

In one aspect, the invention provides a cell containing at least one of the iRNAs (e.g., dsRNAs) featured herein. The cell is generally a mammalian cell, such as a human cell. In some embodiments, the cell is an erythroid cell. In other embodiments, the cell is a liver cell (e.g., a hepatocyte).

In an aspect provided herein is a pharmaceutical composition for inhibiting expression of an ALAS1 gene, the composition comprising an iRNA (e.g., a dsRNA) described herein.

In embodiments of the pharmaceutical compositions described herein, the iRNA (e.g., dsRNA) is administered in an unbuffered solution. In embodiments, the unbuffered solution is saline or water.

In embodiments of the pharmaceutical compositions described herein, the iRNA (e.g., dsRNA is administered with a buffer solution. In embodiments, the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof. In embodiments, the buffer solution is phosphate buffered saline (PBS).

In embodiments of the pharmaceutical compositions described herein, the iRNA (e.g., dsRNA) is targeted to hepatocytes.

In embodiments of the pharmaceutical compositions described herein, the composition is administered intravenously.

In embodiments of the pharmaceutical compositions described herein, the composition is administered subcutaneously.

In embodiments, a pharmaceutical composition comprises an iRNA (e.g., a dsRNA) described herein that comprises a ligand (e.g., a GalNAc ligand) that targets the iRNA (e.g., dsRNA) to hepatocytes.

In embodiments, a pharmaceutical composition comprises an iRNA (e.g., a dsRNA) described herein that comprises a ligand (e.g., a GalNAc ligand), and the pharmaceutical composition is administered subcutaneously. In embodiments, the ligand targets the iRNA (e.g., dsRNA) to hepatocytes.

In certain embodiments, a pharmaceutical composition, e.g., a composition described herein, includes a lipid formulation. In some embodiments, the RNAi agent is in a LNP formulation, e.g., a MC3 formulation. In some embodiments, the LNP formulation targets the RNAi agent to a particular cell, e.g., a liver cell, e.g., a hepatocyte. In embodiments, the lipid formulation is a LNP11 formulation. In embodiments, the composition is administered intravenously.

In another embodiment, the pharmaceutical composition is formulated for administration according to a dosage regimen described herein, e.g., not more than once every four weeks, not more than once every three weeks, not more than once every two weeks, or not more than once every week. In another embodiment, the administration of the pharmaceutical composition can be maintained for a month or longer, e.g., one, two, three, or six months, or one year or longer.

In another embodiment, a composition containing an iRNA featured in the invention, e.g., a dsRNA targeting ALAS1, is administered with a non-iRNA therapeutic agent, such as an agent known to treat a porphyria (e.g., AIP), or a symptom of a porphyria (e.g., pain). In another embodiment, a composition containing an iRNA featured in the invention, e.g., a dsRNA targeting AIP, is administered along with a non-iRNA therapeutic regimen, such as hemin or glucose (e.g., glucose infusion (e.g., IV glucose)). For example, an iRNA featured in the invention can be administered before, after, or concurrent with glucose, dextrose, or a similar treatment that serves to restore energy balance (e.g., total parenteral nutrition). An iRNA featured in the invention can also be administered before, after, or concurrent with the administration of a heme product (e.g., hemin, heme arginate, or heme albumin), and optionally also in combination with a glucose (e.g. IV glucose) or the like.

Typically, glucose administered for the treatment of a porphyria is administered intravenously (IV). Administration of glucose intravenously is referred to herein as “IV glucose.” However, alternative embodiments in which glucose is administered by other means are also encompassed.

In one embodiment, an ALAS1 iRNA is administered to a patient, and then the non-iRNA agent or therapeutic regimen (e.g., glucose and/or a heme product) is administered to the patient (or vice versa). In another embodiment, an ALAS1 iRNA and the non-iRNA therapeutic agent or therapeutic regimen are administered at the same time.

In an aspect provided herein is a method of inhibiting ALAS1 expression in a cell, the method comprising: (a) introducing into the cell an iRNA (e.g. a dsRNA) described herein and (b) maintaining the cell of step (a) for a time sufficient to obtain degradation of the mRNA transcript of an ALAS1 gene, thereby inhibiting expression of the ALAS1 gene in the cell.

In an aspect provided herein is a method for reducing or inhibiting the expression of an ALAS1 gene in a cell (e.g., an erythroid cell or a liver cell, such as, e.g., a hepatocyte). The method includes:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid         (dsRNA), wherein the dsRNA includes at least two sequences that         are complementary to each other. The dsRNA has a sense strand         having a first sequence and an antisense strand having a second         sequence; the antisense strand has a region of complementarity         that is substantially complementary to at least a part of an         mRNA encoding ALAS1, and where the region of complementarity is         30 nucleotides or less, i.e., 15-30 nucleotides in length, and         generally 19-24 nucleotides in length, and where the dsRNA upon         contact with a cell expressing ALAS1, inhibits expression of an         ALAS1 gene by at least 10%, e.g., at least 20%, at least 30%, at         least 40% or more; and     -   (b) maintaining the cell of step (a) for a time sufficient to         obtain degradation of the mRNA transcript of the ALAS1 gene,         thereby reducing or inhibiting expression of an ALAS1 gene in         the cell.

In embodiments of the foregoing methods of inhibiting ALAS1 expression in a cell, the cell is treated ex vivo, in vitro, or in vivo. In embodiments, the cell is a hepatocyte.

In embodiments, the cell is present in a subject in need of treatment, prevention and/or management of a disorder related to ALAS1 expression.

In embodiments, the disorder is a porphyria. In embodiments, the porphyria is acute intermittent porphyria or ALA-dehydratase deficiency porphyria.

In embodiments, the porphyria is a hepatic porphyria, e.g., a porphyria selected from acute intermittent porphyria (AIP) hereditary coproporphyria (HCP), variegate porphyria (VP), ALA deyhdratase deficiency porphyria (ADP), and hepatoerythropoietic porphyria. In embodiments, the porphyria is a homozygous dominant hepatic porphyria (e.g., homozygous dominant AIP, HCP, or VP) or hepatoerythropoietic porphyria, In embodiments, the porphyria is a dual porphyria.

In embodiments, the expression of ALAS1 is inhibited by at least 30%.

In embodiments, the iRNA (e.g., dsRNA) has an IC₅₀ in the range of 0.01-1 nM.

In certain embodiments, the cell (e.g., the hepatocyte) is a mammalian cell (e.g., a human, non-human primate, or rodent cell).

In one embodiment, the cell is treated ex vivo, in vitro, or in vivo (e.g., the cell is present in a subject (e.g., a patient in need of treatment, prevention and/or management of a disorder related to ALAS1 expression).

In one embodiment, the subject is a mammal (e.g., a human) at risk, or diagnosed with a porphyria, e.g., X-linked sideroblastic anemia (XLSA), ALA deyhdratase deficiency porphyria (ADP or Doss porphyria), acute intermittent porphyria (AIP), congenital erythropoietic porphyria (CEP), prophyria cutanea tarda (PCT), hereditary coproporphyria (coproporphyria, or HCP), variegate porphyria (VP), erythropoietic protoporphyria (EPP), or transient erythroporphyria of infancy. In some embodiments, the disorder is an acute hepatic porphyria, e.g., ALA deyhdratase deficiency porphyria (ADP), AIP, HCP, or VP. In specific embodiments, the disorder is ALA deyhdratase deficiency porphyria (ADP) or AIP.

In embodiments, the porphyria is a hepatic porphyria, e.g., a porphyria selected from acute intermittent porphyria (AIP) hereditary coproporphyria (HCP), variegate porphyria (VP), ALA deyhdratase deficiency porphyria (ADP), and hepatoerythropoietic porphyria. In embodiments, the porphyria is a homozygous dominant hepatic porphyria (e.g., homozygous dominant AIP, HCP, or VP) or hepatoerythropoietic porphyria, In embodiments, the porphyria is a dual porphyria.

In one embodiment, the dsRNA introduced reduces or inhibits expression of an ALAS1 gene in the cell.

In one embodiment, the dsRNA introduced reduces or inhibits expression of an ALAS1 gene, or the level of one or more porphyrins or porphyrin precursors (e.g., δ-aminolevulinic acid (ALA), porphopilinogen (PBG), hydroxymethylbilane (HMB), uroporphyrinogen I or III, coproporphyrinogen I or III, protoporphrinogen IX, and protoporphyrin IX) or porphyrin products or metabolites, by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more compared to a reference, (e.g., an untreated cell or a cell treated with a non-targeting control dsRNA). Without being bound by theory, ALAS1 is the first enzyme of the porphyrin pathway. Thus, reducing expression of the ALAS1 gene is likely to reduce the level of one or more porphyrin precursors, porphyrins or porphyrin products or metabolites.

In other aspects, the invention provides methods for treating, preventing or managing pathological processes related to ALAS1 expression (e.g., pathological processes involving porphyrins, porphyrin precuorsors, or defects in the porphyrin pathway, such as, for example, porphyrias). In one embodiment, the method includes administering to a subject, e.g., a patient in need of such treatment, prevention or management, an effective (e.g., a therapeutically or prophylactically effective) amount of one or more of the iRNAs featured herein.

In an aspect provided herein is a method of treating and/or preventing a disorder related to ALAS1 expression comprising administering to a subject in need of such treatment a therapeutically effective amount of an iRNA (e.g., a dsRNA) described herein, or a composition comprising an iRNA (e.g., a dsRNA) described herein.

In an aspect provided herein is a method of treating and/or preventing a porphyria comprising administering to a subject in need of such treatment a double-stranded ribonucleic acid (dsRNA), wherein said dsRNA comprises a sense strand and an antisense strand 15-30 base pairs in length and the antisense strand is complementary to at least 15 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:382.

In one embodiment, subject (e.g., the patient) has a porphyria. In another embodiment, the subject (e.g., patient) is at risk for developing a porphyria. In some embodiments, administration of the iRNA targeting ALAS1 alleviates or relieves the severity of at least one symptom of a disorder related to ALAS1 in the patient.

In one embodiment, the subject is a mammal (e.g., a human) at risk, or that has been diagnosed with, a disorder related to ALAS1 expression, e.g., a porphyria, e.g., X-linked sideroblastic anemia (XLSA), ALA deyhdratase deficiency porphyria (Doss porphyria), acute intermittent porphyria (AIP), congenital erythropoietic porphyria (CEP), prophyria cutanea tarda (PCT), hereditary coproporphyria (coproporphyria, or HCP), variegate porphyria (VP), erythropoietic protoporphyria (EPP), or transient erythroporphyria of infancy. In a further embodiment, the porphyria is an acute hepatic porphyria, e.g., ALA deyhdratase deficiency porphyria (ADP), AIP, HCP, or VP. In some such embodiments, the disorder is ALA deyhdratase deficiency porphyria (ADP) or AIP.

In embodiments the subject has, or is at risk for developing, a porphyria. In embodiments, the porphyria is a hepatic porphyria, e.g., a porphyria selected from acute intermittent porphyria (AIP) hereditary coproporphyria (HCP), variegate porphyria (VP), ALA deyhdratase deficiency porphyria (ADP), and hepatoerythropoietic porphyria. In embodiments, the porphyria is a homozygous dominant hepatic porphyria (e.g., homozygous dominant AP, HCP, or VP) or hepatoerythropoietic porphyria, In embodiments, the porphyria is a dual porphyria.

In embodiments, a porphyria, a symptom of porphyria, a prodrome, or an attack of porphyria is induced by exposure to a precipitating factor, as described herein. In some embodiments, the precipitating factor is a chemical exposure. In some embodiments, the precipitating factor is a drug, e.g., a prescription drug or an over the counter drug. In some embodiments, the precipitating factor is the menstrual cycle, e.g., a particular phase of the menstrual cycle, e.g., the luteal phase.

In embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered after an acute attack of porphyria.

In embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered during an acute attack of porphyria.

In embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered prophylactically to prevent an acute attack of porphyria.

In embodiments, the iRNA (e.g., dsRNA) is formulated as an LNP formulation.

In embodiments, the iRNA (e.g., dsRNA) is in the form of a GalNAc conjugate.

In embodiments, iRNA (e.g., dsRNA) is administered at a dose of 0.05-50 mg/kg.

In embodiments, the iRNA (e.g., dsRNA) is administered at a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the subject.

In embodiments, the iRNA (e.g., dsRNA) is formulated as an LNP formulation and is administered at a dose of 0.05-5 mg/kg.

In embodiments, the iRNA (e.g., dsRNA) is in the form of a GalNAc conjugate and is administered at a dose of 0.5-50 mg/kg.

In embodiments, the method decreases a level of a porphyrin or a porphyrin precursor in the subject.

In embodiments, the level is decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In an embodiment, the level is decreased by at least 30%.

In embodiments, the porphyrin precursor is δ-aminolevulinic acid (ALA) or porphopilinogen (PBG).

In embodiments, the iRNA (e.g., dsRNA) has an IC₅₀ in the range of 0.01-1 nM.

In embodiments, a method described herein

-   -   (i) ameliorates a symptom associated with an ALAS1 related         disorder (e.g., a porphyria)     -   (ii) inhibits ALAS1 expression in the subject,     -   (iii) decreases a level of a porphyrin precursor (e.g., ALA or         PBG) or a porphyrin in the subject,     -   (iv) decreases frequency of acute attacks of symptoms associated         with a porphyria in the subject, or     -   (v) decreases incidence of acute attacks of symptoms associated         with a porphyria in the subject when the subject is exposed to a         precipitating factor (e.g., the premenstrual phase or the luteal         phase).

In embodiments, the method ameliorates pain and/or progressive neuropathy.

In embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered according to a dosing regimen.

In some embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered before or during an acute attack of porphyria. In some embodiments, the iRNA is administered before an acute attack of porphyria.

In some embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered during a prodrome. In embodiments, the prodrome is characterized by abdominal pain, nausea, psychological symptoms (e.g., anxiety), restlessness and/or insomnia.

In embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered during a particular phase of the menstrual cycle, e.g., during the luteal phase.

In embodiments, the method ameliorates or prevents cyclical attacks of porphyria, e.g., by reducing the severity, duration, or frequency of attacks. In embodiments, the cyclical attacks are associated with a precipitating factor. In embodiments, the precipitating factor is the menstrual cycle, e.g., a particular phase of the menstrual cycle, e.g., the luteal phase.

In embodiments, the subject has an elevated level of ALA and/or PBG. In embodiments, the subject has or is at risk for developing a porphyria, e.g., a hepatic porphyria. In embodiments, the subject is asymptomatic. In embodiments, the subject carries a genetic alteration (e.g., a gene mutation) associated with a porphyria, as described herein.

In embodiments, the subject has or is at risk for developing a porphyria and suffers from pain (e.g., chronic pain, e.g., chronic neuropathic pain) and/or neuropathy (e.g., progressive neuropathy). In embodiments, the subject does not suffer from acute attacks but suffers from pain (e.g., chronic pain, e.g., chronic neuropathic pain) and/or neuropathy (e.g., progressive neuropathy). In embodiments, the pain is abdominal pain.

In embodiments, the subject (a) has an elevated level of ALA and/or PBG and (b) suffers from pain (e.g., chronic pain, e.g., chronic neuropathic pain) and/or neuropathy (e.g., progressive neuropathy). In embodiments, the pain is abdominal pain.

In embodiments, the subject has a plasma level and/or a urine level of ALA and/or PBG that is elevated. In embodiments, the elevated level of ALA and/or PBG is accompanied by other symptoms, e.g., pain (e.g., chronic pain, e.g., chronic neuropathic pain) or neuropathy (e.g., progressive neuropathy). In embodiments, the pain is abdominal pain. In embodiments, the subject is asymptomatic. In embodiments, the subject has a genetic mutation associated with a porphyria, e.g., a mutation as described herein.

In embodiments, the subject has a level (e.g., a plasma level or a urine level) of a porphyrin precursor, e.g., ALA and/or PBG, that is elevated, e.g., the level is greater than, or greater than or equal to, a reference value. In embodiments, the level is greater than the reference value. In embodiments, the reference value is two standard deviations above the mean level in a sample of healthy individuals. In embodiments, the reference value is an upper reference limit.

In embodiments, the subject has a plasma level and/or a urine level of ALA and/or PBG that is greater than, or greater than or or equal to, 2 times, 3 times, 4 times, or 5 times that of an upper reference limit. As used herein, an “upper reference limit” refers to a level that is the upper limit of the 95% confidence interval for a reference sample, e.g., a sample of normal (e.g., wild type) or healthy individuals, e.g., individuals who do not carry a genetic mutation associated with a porphyria and/or individuals who do not suffer from a porphyria. In embodiments, the subject has a urine level of ALA and/or PBG that is greater than 2 to 4 times that of an upper reference limit. In embodiments, the subject has a urine level of ALA and/or PBG that is greater than 4 times that of an upper reference limit.

In embodiments, the reference value for plasma PBG is 0.12 μmol/L. In embodiments, the subject is a human and has a plasma PBG level that is greater than, or greater than or equal to, 0.12 μmol/L, 0.24 μmol/L, 0.36 μmol/L, 0.48 μmol/L, or 0.60 μmol/L. In embodiments, the subject is a human and has a plasma level of PBG that is greater than, or greater than or equal to, 0.48 μmol/L.

In embodiments, the reference value for urine PBG is 1.2 mmol/mol creatinine. In embodiments, the subject is a human and has a urine PBG level that is greater than, or greater than or equal to, 1.2 mmol/mol creatinine, 2.4 mmol/mol creatinine, 3.6 mmol/mol creatinine, 4.8 mmol/mol creatinine, or 6.0 mmol/mol creatinine. In embodiments, the subject is a human and has a urine level of PBG that is greater than, or greater than or equal to, 4.8 mmol/mol creatinine.

In embodiments, the reference value for plasma ALA is 0.12 μmol/L. In embodiments, the subject is a human and has a plasma ALA level that is greater than, or greater than or equal to, 0.12 μmol/L, 0.24 μmol/L, 0.36 μmol/L, 0.48 μmol/L, or 0.60 μmol/L. In embodiments, the subject is a human and has a plasma ALA level that is greater than, or greater than or equal to 0.48 μmol/L.

In embodiments, the reference value for urine ALA is 3.1 mmol/mol creatinine. In embodiments, the subject is a human and has a urine ALA level that is greater than, or greater than or equal to, 3.1 mmol/mol creatinine, 6.2 mmol/mol creatinine, 9.3 mmol/mol creatinine, 12.4 mmol/mol creatinine, or 15.5 mmol/mol creatinine.

In embodiments, the method decreases an elevated level of ALA and/or PBG. In embodiments, the method decreases pain (e.g., chronic pain, e.g. chronic neuropathic pain) and/or neuropathy (e.g., progressive neuropathy). In embodiments, the pain is abdominal pain.

In embodiments, the pain is neuropathic pain (e.g., pain associated with the progressive neuropathy of acute porphyrias). The decrease in pain can include, e.g., prevention of pain, delay in the onset of pain, reduction in the frequency of pain, and/or reduction in severity of pain.

In embodiments, the method ameliorates or prevents acute attacks of porphyria, e.g., by reducing the severity, duration, or frequency of attacks.

In embodiments, the method decreases or prevents nerve damage.

In embodiments, the method prevents deterioration (e.g., prevents development of abnormalities) of or results in an improvement of clinical measures, e.g., clinical measures of muscle and/or nerve function, e.g., EMG and/or nerve conduction velocities.

In embodiments, the method is effective to reduce a level of ALA and/or PBG (e.g., a plasma or urine level of ALA and/or PBG). In embodiments, the method is effective to produce a predetermined reduction in the elevated level of ALA and/or PBG.

In embodiments, the predetermined reduction is a reduction to a value that is less than or equal to a reference value. In some embodiments, the reference value is an upper reference limit.

In some embodiments, the reference value is the value that is two standard deviations above the mean level in a reference sample.

In embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered repeatedly, e.g., according to a dosing regimen.

In embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered prophylactically to a subject who is at risk for developing a porphyria. In embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered prophylactically beginning at puberty. In embodiments, the subject carries a genetic mutation associated with a porphyria and/or has an elevated level of ALA and/or PBG (e.g., an elevated plasma or urine level of ALA and/or PBG). In embodiments, the mutation makes an individual susceptible to an acute attack (e.g., upon exposure to a precipitating factor, e.g., a drug, dieting or other precipitating factor, e.g., a precipitating factor as disclosed herein). In embodiments, the mutation is associated with elevated levels of a porphyrin or a porphyrin precursor (e.g., ALA and/or PBG). In embodiments, the mutation is associated with chronic pain (e.g., chronic neuropathic pain) and/or neuropathy (e.g., progressive neuropathy).

In embodiments, the mutation is a mutation in the ALAS1 gene. In embodiments, the mutation is a mutation in the ALAS1 gene promoter, or in regions upstream or downstream from the ALAS1 gene. In embodiments, the mutation is a mutation in transcription factors or other genes that interact with ALAS1. In embodiments, the mutation is a mutation in a gene that encodes an enzyme in the heme biosynthetic pathway.

In embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered subcutaneously. In embodiments, the iRNA is in the form of a GalNAc conjugate. In embodiments, the iRNA (e.g., the dsRNA) is administered at a dose of 0.5-50 mg/kg.

In one aspect provided herein is a method of treating a subject with an elevated level of ALA and/or PBG, the method comprising administering to the subject a double-stranded ribonucleic acid (dsRNA), wherein said dsRNA comprises a sense strand and an antisense strand 15-30 base pairs in length and the antisense strand is complementary to at least 15 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:382.

In one aspect provided herein is a method of treating a subject with an elevated level of ALA and/or PBG, the method comprising administering to the subject a therapeutically effective amount of an dsRNA or a composition comprising a dsRNA, as described herein.

In some embodiments, the methods described herein are effective to decrease the level of ALA and/or PBG. In some embodiments, the level of ALA and/or PBG is decreased such that it is less than, or less than or equal to, a reference value, e.g., an upper reference limit. In another aspect, the invention provides methods for decreasing a level of a porphyrin or a porphyrin precursor in a cell (e.g., an erythroid cell or a liver cell, such as, e.g., a hepatocyte). In one embodiment, the cell is treated ex vivo, in vitro, or in vivo (e.g., the cell is present in a subject (e.g., a patient in need of treatment, prevention and/or management of a disorder related to ALAS1 expression). The method includes contacting the cell with an effective amount of one or more of the iRNAs targeting ALAS1, e.g., one or more of the iRNAs disclosed herein, thereby decreasing the level of a porphyrin or a porphyrin precursor in the cell; or decreasing the level of a porphyrin or a porphyrin precursor in other cells, tissues, or fluids within a subject in which the cell is located; relative to the level prior to contacting. Such methods can be used to treat (e.g., ameliorate the severity) of disorders related to ALAS1 expression, such as porphyrias, e.g., AIP or ALA dehydratase deficiency porphyria.

In one embodiment, the contacting step is effected ex vivo, in vitro, or in vivo. For example, the cell can be present in a subject, e.g., a mammal (e.g., a human) at risk, or that has been diagnosed with, a porphyria. In an embodiment, the porphyria is an acute hepatic porphyria. In embodiments, the porphyria is a hepatic porphyria, e.g., a porphyria selected from acute intermittent porphyria (AIP), hereditary coproporphyria (HCP), variegate porphyria (VP), ALA deyhdratase deficiency porphyria (ADP), and hepatoerythropoietic porphyria. In embodiments, the porphyria is a homozygous dominant hepatic porphyria (e.g., homozygous dominant AIP, HCP, or VP) or hepatoerythropoietic porphyria, In embodiments, the porphyria is a dual porphyria.

In an aspect provided herein is a method for decreasing a level of a porphyrin or a porphyrin precursor (e.g., ALA or PBG) in a cell, comprising contacting the cell with an iRNA (e.g. a dsRNA), as described herein, in an amount effective to decrease the level of the porphyrin or the porphyrin precursor in the cell. In embodiments, the cell is a hepatocyte. In embodiments, the porphyrin or porphyrin precursor is δ-aminolevulinic acid (ALA), porphopilinogen (PBG), hydroxymethylbilane (HMB), uroporphyrinogen I or III, coproporphyrinogen I or III, protoporphrinogen IX, or protoporphyrin IX. In embodiments, the porphyrin precursor is ALA or PBG.

In one embodiment, the cell is an erythroid cell. In a further embodiment, the cell is a liver cell (e.g., a hepatocyte).

In an aspect provided herein is a vector encoding at least one strand of an iRNA (e.g., a dsRNA) as described herein.

In an aspect provided herein is a vector encoding at least one strand of a dsRNA, wherein said dsRNA comprises a region of complementarity to at least a part of an mRNA encoding ALAS1, wherein said dsRNA is 30 base pairs or less in length, and wherein said dsRNA targets said mRNA for cleavage.

In embodiments, the region of complementarity is at least 15 nucleotides in length.

In embodiments, the region of complementarity is 19 to 21 nucleotides in length. In one aspect, the invention provides a vector for inhibiting the expression of an ALAS1 gene in a cell.

In one embodiment, the vector comprises an iRNA as described herein. In one embodiment, the vector includes at least one regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of an iRNA as described herein. In one embodiment the vector comprises at least one strand of an ALAS1 iRNA.

In an aspect provided herein is a cell comprising a vector as described herein. In an aspect provided herein is a cell containing a vector for inhibiting the expression of an ALAS1 gene in a cell. The vector includes a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the iRNAs as described herein. In one embodiment, the cell is a liver cell (e.g., a hepatocyte). In another embodiment, the cell is an erythroid cell.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the heme biosynthetic pathway.

FIG. 2A is a table that summarizes certain porphyrias associated with genetic errors in heme metabolism.

FIG. 2B is a continuation of the table in FIG. 2A.

FIG. 3A depicts nucleotides 1-2280 of the sequence of a human ALAS1 mRNA sequence transcript variant 1 (Ref. Seq. NM_000688.4 (GI:40316942, record dated Nov. 19, 2011), SEQ ID NO: 1).

FIG. 3B depicts nucleotides 2281-2407 of the sequence of a human ALAS1 mRNA sequence transcript variant 1 (Ref. Seq. NM_000688.4 (GI:40316942, record dated Nov. 19, 2011), SEQ ID NO: 1).

FIG. 4A depicts nucleotides 1-2280 of the sequence of a human ALAS1 mRNA sequence transcript variant 2 (Ref. Seq. NM_000688.5 (GI: 362999011, record dated Apr. 1, 2012), SEQ ID NO: 382).

FIG. 4B depicts nucleotides 2281-2458 of the sequence of a human ALAS1 mRNA sequence transcript variant 2 (Ref. Seq. NM_000688.5 (GI: 362999011, record dated Apr. 1, 2012), SEQ ID NO: 382).

FIG. 5 shows the dose-response of the siRNA AD-53558 in suppressing mouse ALAS1 (mALAS1) mRNA relative to a PBS control. Results for a luciferase (LUC) AD-1955 control are also shown.

FIG. 6 shows the dose-response of the siRNA AD-53558 in suppressing ALAS1 mRNA in rats relative to a PBS control. Results for a luciferase (LUC) AD-1955 control are also shown.

FIG. 7 shows the durability of suppression of mouse ALAS1 (mALAS1) mRNA by the siRNA AD-53558 relative to a PBS control.

FIG. 8 shows means standard deviations of plasma ALA levels (in μM) at baseline, and after phenobarbitol treatment in the experimental (ALAS1 siRNA) and control (LUC siRNA) groups.

FIG. 9 shows shows the plasma ALA levels (in μM) of individual animals at baseline, and after phenobarbitol treatment in animals that received ALAS1 siRNA and control (LUC siRNA) treatment.

FIG. 10 shows means standard deviations of plasma PBG levels (in μM) at baseline, and after phenobarbitol treatment in animals that received ALAS1 siRNA and control (LUC siRNA) treatment.

FIG. 11 shows shows the plasma PBG levels (in μM) of individual animals at baseline, and after phenobarbitol treatment in animals that received ALAS1 siRNA and control (LUC siRNA) treatment.

FIG. 12 shows the relative mALAS1 mRNA level in liver at baseline, and after phenobarbitol treatment in select representative experimental (ALAS1 siRNA) and control (PBS) animals.

FIG. 13 shows the effects of three GalNAc conjugated mALAS1 siRNAs on mALAS1 expression (relative to a PBS control) in mouse liver tissue.

FIG. 14 shows plasma ALA and PBG levels over time after phenobarbitol administration and treatment with ALAS1 siRNA or control LUC siRNA.

DETAILED DESCRIPTION OF THE INVENTION

iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). Described herein are iRNAs and methods of using them for inhibiting the expression of an ALAS1 gene in a cell or a mammal where the iRNA targets an ALAS1 gene. Also provided are compositions and methods for disorders related to ALAS1 expression, such as porphyrias (e.g., ALA deyhdratase deficiency porphyria (ADP or Doss porphyria), acute intermittent porphyria, congenital erythropoietic porphyria, prophyria cutanea tarda, hereditary coproporphyria (coproporphyria), variegate porphyria, erythropoietic protoporphyria (EPP), X-linked sideroblastic anemia (XLSA), and and transient erythroporphyria of infancy).

Porphyrias are inherited or acquired disorders that can be caused by decreased or enhanced activity of specific enzymes in the heme biosynthetic pathway, also referred to herein as the porphyrin pathway (See FIG. 1). Porphyrins are the main precursors of heme. Porphyrins and porphyrin precursors include δ-aminolevulinic acid (ALA), porphopilinogen (PBG), hydroxymethylbilane (HMB), uroporphyrinogen I or III, coproporphyrinogen I or III, protoporphrinogen IX, and protoporphyrin IX. Heme is an essential part of hemoglobin, myoglobin, catalases, peroxidases, and cytochromes, the latter including the respiratory and P450 liver cytochromes. Heme is synthesized in most or all human cells. About 85% of heme is made in erythroid cells, primarily for hemoglobin. Most of the remaining heme is made in the liver, 80% of which is used for the synthesis of cytochromes. Deficiency of specific enzymes in the porphyrin pathway leads to insufficient heme production and also to an accumulation of porphyrin precursors and/or porphyrins, which can be toxic to cell or organ function in high concentrations.

Porphyrias may manifest with neurological complications (“acute”), skin problems (“cutaneous”) or both. Porphyrias may be classified by the primary site of the overproduction and accumulation of porphyrins or their precursors. In hepatic porphyrias, porphyrins and porphyrin precursors are overproduced predominantly in the liver, whereas in erythropoietic porphyrias, porphyrins are overproduced in the erythroid cells in the bone. The acute or hepatic porphyrias lead to dysfunction of the the nervous system and neurologic manifestations that can affect both the central and peripheral nervous system, resulting in symptoms such as, for example, pain (e.g., abdominal pain and/or chronic neuropathic pain), vomiting, neuropathy (e.g., acute neuropathy progressive neuropathy), muscle weakness, seizures, mental disturbances (e.g., hallucinations, depression anxiety, paranoia), cardiac arrhythmias, tachycardia, constipation, and diarrhea. The cutaneous or erythropoietic porphyrias primarily affect the skin, causing symptoms such as photosensitivity that can be painful, blisters, necrosis, itching, swelling, and increased hair growth on areas such as the forehead. Subsequent infection of skin lesions can lead to bone and tissue loss, as well as scarring, disfigurement, and loss of digits (e.g., fingers, toes). Most porphyrias are caused by mutations that encode enzymes in the heme biosynthetic pathway. A summary of porphyrias associated with genetic errors in heme metabolism is provided in FIG. 2.

Not all porphyrias are genetic. For example, patients with liver disease may develop porphyria as a result of liver dysfunction, and a transient form of erythroporphria (transient erythroporphyria of infancy) has been described in infancy (see Crawford, R. I. et al, J Am Acad Dermatol. 1995 August; 33(2 Pt 2):333-6.) Patients with PCT can acquire the deficient activity of uroporphyrinogen decarboxylase (URO-D), due to the formation of a ORO-D enzyme with lower than normal enzymatic activity (see Phillips et al. Blood, 98:3179-3185, 2001.)

Acute intermittent porphyria (AIP) (also be referred to as porphobilinogen (PBG) deaminase deficiency, or hydroxymethylbilane synthase (HMBS) deficiency), is the most common type of acute hepatic porphyria. Other types of acute hepatic porphyrias include hereditary coproporphyria (HCP), variegate porphyria (VP), and ALA deyhdratase deficiency porphyria (ADP). Acute hepatic porphyrias are described, e.g., in Balwani, M and Desnick, R. J., Blood, 120:4496-4504, 2012.

AIP is typically an autosomal dominant disease that is characterized by a deficiency of the enzyme porphobilinogen deaminase (PBG deaminase); this enzyme is also known as hydroxymethylbilane synthase (HMB synthase or HMBS). PBG deaminase is the third enzyme of the heme biosynthetic pathway (see FIG. 1) and catalyzes the head to tail condensation of four porphobilinogen molecules into the linear tetrapyrrole, hydroxymethylbilane (HMB). Alternatively spliced transcript variants encoding different isoforms of PBG deaminase have been described. Mutations in the PBG deaminase gene are associated with AP. Such mutations may lead to decreased amounts of PBG deaminase and/or decreased activity of PBG deaminase (affected individuals typically have a 50% reduction in PBG deaminase activity).

There are at least two different models of the pathophysiology of AP and other acute hepatic porphyrias (see, e.g., Lin CS-Y et al., Clinical Neurophysiology, 2011; 122:2336-44). According to one model, the decreased heme production resulting from PBG deaminase deficiency causes energy failure and axonal degeneration. According to the other, currently more favored model, the buildup of porphyrin precursors (e.g., ALA and PBG) results in neurotoxicity.

AIP has been found to have a prevalence as high as 1 in 10,000 in certain populations (e.g., in Northern Sweden; see Floderus Y, et al. Clin Genet. 2002; 62:288-97). The prevalence in the general population in United States and Europe, excluding the U.K., is estimated to be about 1 in 10,000 to 1 in 20,000. Clinical disease manifests itself in only approximately 10-15% of individuals who carry mutations that are known to be associated with AP. However, the penetrance is as high as 40% in individuals with certain mutations (e.g., the W198X mutation). AIP is typically latent prior to puberty. Symptoms are more common in females than in males. The prevalence of the disease is probably underestimated due to its incomplete penetrance and long periods of latency. In the United States, it is estimated that there are about 2000 patients who have suffered at least one attack. It is estimated that there are about 150 active recurrent cases in France, Sweden, the U.K., and Poland; these patients are predominantly young women, with a median age of 30. See, e.g., Elder et al, J Inherit Metab Dis., published online Nov. 1, 2012.

AIP affects, for example, the visceral, peripheral, autonomic, and central nervous systems. Symptoms of AIP are variable and include gastrointestinal symptoms (e.g., severe and poorly localized abdominal pain, nausea/vomiting, constipation, diarrhea, ileus), urinary symptoms (dysuria, urinary retention/incontinence, or dark urine), neurologic symptoms (e.g., sensory neuropathy, motor neuropathy (e.g., affecting the cranial nerves and/or leading to weakness in the arms or legs), seizures, neuropathic pain (e.g., pain associated with progressive neuropathy, e.g., chronic neuropathic pain), neuropsychiatric symptoms (e.g., mental confusion, anxiety, agitation, hallucination, hysteria, delirium, apathy, depression, phobias, psychosis, insomnia, somnolence, coma), autonomic nervous system involvement (resulting e.g., in cardiovascular symptoms such as tachycardia, hypertension, and/or arrhythmias, as well as other symptoms, such as, e.g., increased circulating catecholamine levels, sweating, restlessness, and/or tremor), dehydration, and electrolyte abnormalities. The most common symptoms are abdominal pain and tachycardia. In addition, patients frequently have chronic neuropathic pain and develop a progressive neuropathy. Patients with recurring attacks often have a prodrome. Permanent paralysis may occur after a severe attack. Recovery from severe attacks that are not promptly treated may take weeks or months. An acute attack may be fatal, for example, due to paralysis of respiratory muscles or cardiovascular failure from electrolyte imbalance. (See, e.g., Thunell S. Hydroxymethylbilane Synthase Deficiency. 2005 Sep. 27 [Updated 2011 Sep. 1]. In: Pagon R A, Bird T D, Dolan C R, et al., editors. GeneReviews™ [Internet]. Seattle (Wash.): University of Washington, Seattle; 1993—(hereinafter Thunell (1993)), which is hereby incorporated by reference in its entirety.) Prior to the availability of Hemin treatments, up to 20% of patients with AIP died from the disease.

In individuals who carry genes for AIP, the risk of hepatocellular cancer is increased. In those with recurrent attacks, the risk of hepatocellular cancer is particularly grave: after the age of 50, the risk is nearly 100-fold greater than in the general population.

Attacks of acute porphyria may be precipitated by endogenous or exogenous factors. The mechanisms by which such factors induce attacks may include, for example, increased demand for hepatic P450 enzymes and/or induction of ALAS1 activity in the liver. Increased demand for hepatic P450 enzymes results in decreased hepatic free heme, thereby inducing the synthesis of hepatic ALAS1.

Precipitating factors include fasting (or other forms of reduced or inadequate caloric intake, due to crash diets, long-distance athletics, etc.), metabolic stresses (e.g., infections, surgery, international air travel, and psychological stress), endogenous hormones (e.g., progesterone), cigarette smoking, lipid-soluble foreign chemicals (including, e.g., chemicals present in tobacco smoke, certain prescription drugs, organic solvents, biocides, components in alcoholic beverages), endocrine factors (e.g., reproductive hormones (women may experience exacerbations during the premenstrual period), synthetic estrogens, progesterones, ovulation stimulants, and hormone replacement therapy). See, for example, Thunell (1993).

Over 1000 drugs are contraindicated in the acute hepatic porphyrias (e.g., AIP, HCP, ADP, and VP) including, for example, alcohol, barbiturates, Carbamazepine, Carisoprodol, Clonazepam (high doses), Danazol, Diclofenac and possibly other NSAIDS, Ergots, estrogens, Ethyclorvynol, Glutethimide, Griseofulvin, Mephenytoin, Meprobamate (also mebutamate and tybutamate), Methyprylon, Metodopramide, Phenytoin, Primidone, progesterone and synthetic progestins, Pyrazinamide, Pyrazolones (aminopyrine and antipyrine), Rifampin, Succinimides (ethosuximide and methsuximide), sulfonamide antibiotics, and Valproic acid.

Objective signs of AIP include discoloration of the urine during an acute attack (the urine may appear red or red-brown), and increased concentrations of PBG and ALA in urine during an acute attack. Molecular genetic testing identifies mutations in the PBG deaminase (also known as HMBS) gene in more than 98% of affected individuals. Thunell (1993).

The differential diagnosis of porphyrias may involve determining the type of porphyria by measuring individual levels of porphyrins or porphyrin precursors (e.g., ALA, PBG) in the urine, feces, and/or plasma (e.g., by chromatography and fluorometry) during an attack. The diagnosis of AIP can be confirmed by establishing that erythrocyte PBG deaminase activity is at 50% or less of the normal level. DNA testing for mutations may be carried out in patients and at-risk family members. The diagnosis of AIP is typically confirmed by DNA testing to identify a specific caustative gene mutation (e.g., an HMBS mutation).

Treatment of acute attacks typically requires hospitalization to control and treat acute symptoms, including, e.g., abdominal pain, seizures, dehydration/hyponatremia, nausea/vomiting, tachycardia/hypertension, urinary retention/ileus. For example, abdominal pain may be treated, e.g., with narcotic analgesics, seizures may be treated with seizure precautions and possibly medications (although many anti-seizure medications are contraindicated), nausea/vomiting may be treated, e.g., with phenothiazines, and tachycardia/hypertension may be treated, e.g., with beta blockers. Treatment may include withdrawal of unsafe medications, monitoring of respiratory function, as well as muscle strength and neurological status. Mild attacks (e.g., those with no paresis or hyponatremia) may be treated with at least 300 g intravenous 10% glucose per day, although increasingly hemin is provided immediately. Severe attacks should be treated as soon as possible with intravenous hemin (3-4 mg/kg daily for 4-14 days) and with IV glucose while waiting for the IV hemin to take effect. Typically, attacks are treated with IV hemin for 4 days and with IV glucose while waiting for administration of the IV hemin.

Hemin (Panhematin® or hemin for injection, previously known as hematin) is the only heme product approved for use in the United States and was the first drug approved under the Orphan Drug Act. Panhematin® is hemin derived from processed red blood cells (PRBCs), and is Protoporphyrin IX containing a ferric iron ion (Heme B) with a chloride ligand. Heme acts to limit the hepatic and/or marrow synthesis of porphyrin. The exact mechanism by which hemin produces symptomatic improvement in patients with acute episodes of the hepatic porphyrias has not been elucidated; however, its action is likely due to the (feedback) inhibition of δ-aminolevulinic acid (ALA) synthase, the enzyme which limits the rate of the porphyrin/heme biosynthetic pathway. See Panhematin® product label, Lundbeck, Inc., October 2010. Inhibition of ALA synthase should result in reduced production of ALA and PBG as well as porphyrins and porphyrin intermediates.

Drawbacks of hemin include its delayed impact on clinical symptoms and its failure to prevent the recurrence of attacks. Adverse reactions associated with hemin administration may include thrombophlebitis, anticoagulation, thrombocytopenia, renal shut down, or iron overload, which is particularly likely in patients requiring multiple courses of hemin treatment for recurrent attacks. To prevent phlebitis, an indwelling venous catheter is needed for access in patients with recurrent attacks. Uncommonly reported side effects include fever, aching, malaise, hemolysis, anaphalaxis, and circulatory collapse. See Anderson, K. E., Approaches to Treatment and Prevention of Human Porphyrias, in The Porphyrin Handbook: Medical Aspects of Porphyrins, Edited by Karl M. Kadish, Kevin M. Smith, Roger Guilard (2003) (hereinafter Anderson).

Heme is difficult to prepare in a stable form for intravenous administration. It is insoluble at neutral pH but can be prepared as heme hydroxide at pH 8 or higher. Anderson. Panhematin is a lyophilized hemin preparation. When lyophilized hemin is solubilized for intravenous administration, degradation products form rapidly; these degradation products are responsible for a transient anticoagulant effect and for phlebitis at the site of infusion. Anderson. Heme albumin and heme arginate (Normosang, the European version of hemin) are more stable and may potentially cause less thrombophlebitis. However, heme arginate is not approved for use in the United States. Panhemin may be stabilized by solubilizing it for infusion in 30% human albumin rather than in sterile water; however, albumin adds intravascular volume-expanding effects and increases the cost of treatment as well as risk of pathogens since it is isolated from human blood. See, e.g., Anderson.

The successful treatment of an acute attack does not prevent or delay recurrence. There is a question of whether hemin itself can trigger recurring attacks due to induction of heme oxygenase. Nonetheless, in some areas (especially France), young women with multiply recurrent attacks are being treated with weekly hemin with the goal of achieving prophylaxis.

Limited experience with liver transplantation suggests that if successful, it is an effective treatment for AIP. There have been approximately 12 transplants in Europe in human patients, with curative or varying effects. Liver transplantation can restore normal excretion of ALA and PBG and prevent acute attacks. See, e.g., Dar, F. S. et al. Hepatobiliary Pancreat. Dis. Int., 9(1):93-96(2010). Furthermore, if the liver of a patient with AP is transplanted into another patient (“domino transplant”), the patient receiving the transplant may develop AIP.

Among the long-term clinical effects of acute porphyrias is chronic neuropathic pain that may result from a progressive neuropathy due to neurotoxic effects, e.g., of elevated porphyrin precursors (e.g., ALA and/or PBG). Patients may suffer from neuropathic pain prior to or during an acute attack. Older patients may experience increased neuropathic pain with age for which various narcotic drugs are typically prescribed. Electromyogram abnormalities and decreased conduction times have been documented in patients with acute hepatic porphyrias. Of note, untreated, uninduced mice with AIP (PBG deaminase deficiency) develop a progressive motor neuropathy that has been shown to cause progressive quadriceps nerve axon degeneration and loss presumably due to constitutively elevated porphyrin precursor (ALA & PBG) levels, porphyrins and/or heme deficiency (Lindberg et al., J. Clin. Invest., 103(8): 1127-1134, 1999). In patients with acute porphyria (e.g., ADP, AIP, HCP, or VP), levels of porphyrin precursors (ALA & PBG) are often elevated in asymptomatic patients and in symptomatic patients between attacks. Thus, reduction of the porphyrin precursors and resumption of normal heme biosynthesis by reducing the level of ALAS1 expression and/or activity is expected to prevent and/or minimize development of chronic and progressive neuropathy. Treatment, e.g., chronic treatment (e.g., periodic treatment with iRNA as described herein, e.g., treatment according to a dosing regimen as described herein, e.g., weekly or biweekly treatment) can continuously reduce the ALAS1 expression in acute porphyria patients who have elevated levels of porphyrin precursors, porphyrins, porphyrin products or their metabolites. Such treatment may be provided as needed to prevent or reduce the frequency or severity of an individual patient's symptoms (e.g., pain and/or neuropathy) and/or to reduce a level of a porphyrin precursor, porphyrin, porphyrin product or metabolite.

The need exists for identifying novel therapeutics that can be used for the treatment of porphyrias. As discussed above, existing treatments such as hemin have numerous drawbacks. For example, the impact of hemin on clinical symptoms is delayed, it is expensive, and it may have side effects (e.g., thrombophlebitis, anticoagulation, thrombocytopenia, iron overload, renal shutdown). Novel therapeutics such as those described herein can address these drawbacks and the unmet needs of patients by, for example, acting faster, not inducing phlebitis, providing the convenience of subcutaneous administration, successfully preventing recurrent attacks, preventing or ameliorating pain (e.g., chronic neuropathic pain) and/or progressive neuropathy, and/or not causing certain adverse effects associated with hemin (e.g., iron overload, increased risk of hepatocellular cancer).

The present disclosure provides methods and iRNA compositions for modulating the expression of an ALAS1 gene. In certain embodiments, expression of ALAS1 is reduced or inhibited using an ALAS1-specific iRNA, thereby leading to a decreased expression of an ALAS1 gene. Reduced expression of an ALAS1 gene may reduce the level of one or more porphyrin precursors, porphyrins, or porphyrin products or metabolites. Decreased expression of an ALAS1 gene, as well as related decreases in the level of one or more porphyrin precursors and/or porphyrins, can be useful in treating disorders related to ALAS1 expression, e.g., porphyrias.

The iRNAs of the compositions featured herein include an RNA strand (the antisense strand) having a region which is 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of an ALAS1 gene (also referred to herein as an “ALAS1-specific iRNA”). The use of such an iRNA enables the targeted degradation of mRNAs of genes that are implicated in pathologies associated with ALAS1 expression in mammals, e.g., porphyrias such as ALA dehydratase deficiency porphyria (Doss porphyria) or acute intermittent porphyria. Very low dosages of ALAS1-specific iRNAs can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of an ALAS1 gene. iRNAs targeting ALAS1 can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of an ALAS1 gene, e.g., in cell based assays. Thus, methods and compositions including these iRNAs are useful for treating pathological processes related to ALAS1 expression, such as porphyrias (e.g., X-linked sideroblastic anemia (XLSA), ALA deyhdratase deficiency porphyria (Doss porphyria), acute intermittent porphyria (AIP), congenital erythropoietic porphyria, prophyria cutanea tarda, hereditary coproporphyria (coproporphyria), variegate porphyria, erythropoietic protoporphyria (EPP), and transient erythroporphyria of infancy).

The following description discloses how to make and use compositions containing iRNAs to inhibit the expression of an ALAS1 gene, as well as compositions and methods for treating diseases and disorders caused by or modulated by the expression of this gene. Embodiments of the pharmaceutical compositions featured in the invention include an iRNA having an antisense strand comprising a region which is 30 nucleotides or less in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part of an RNA transcript of an ALAS1 gene, together with a pharmaceutically acceptable carrier. Embodiments of compositions featured in the invention also include an iRNA having an antisense strand having a region of complementarity which is 30 nucleotides or less in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of an ALAS1 gene.

Accordingly, in some aspects, pharmaceutical compositions containing an ALAS1 iRNA and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of an ALAS1 gene, and methods of using the pharmaceutical compositions to treat disorders related to ALAS1 expression are featured in the invention.

I. Definitions

For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

As used herein, “ALAS1” (also known as ALAS-1; δ-aminolevulinate synthase 1; δ-ALA synthase 1; 5′-aminolevulinic acid synthase 1; ALAS-H; ALASH; ALAS-N; ALAS3; EC2.3.1.37; 5-aminolevulinate synthase, nonspecific, mitochondrial; ALAS; MIG4; OTTHUMP00000212619; OTTHUMP00000212620; OTTHUMP00000212621; OTTHUMP00000212622; migration-inducing protein 4; EC 2.3.1) refers to a nuclear-encoded mitochondrial enzyme that is the first and typically rate-limiting enzyme in the mammalian heme biosynthetic pathway. ALAS1 catalyzes the condensation of glycine with succinyl-CoA to form S-aminolevulinic acid (ALA). The human ALAS1 gene is expressed ubiquitously, is found on chromosome 3p21.1 and typically encodes a sequence of 640 amino acids. In contrast, the ALAS-2 gene, which encodes an isozyme, is expressed only in erythrocytes, is found on chromoxome Xp11.21, and typically encodes a sequence of 550 amino acids. As used herein an “ALAS1 protein” means any protein variant of ALAS1 from any species (e.g., human, mouse, non-human primate), as well as any mutants and fragments thereof that retain an ALAS1 activity. Similarly, an “ALAS1 transcript” refers to any transcript variant of ALAS1, from any species (e.g., human, mouse, non-human primate). A sequence of a human ALAS1 variant 1 mRNA transcript can be found at NM_000688.4 (FIG. 3; SEQ ID NO:1). Another version, a human ALAS1 variant 2 mRNA transcript, can be found at NM_000688.5 (FIG. 4; SEQ ID NO:382). The level of the mature encoded ALAS1 protein is regulated by heme: high levels of heme down-regulate the mature enzyme in mitochondria while low heme levels up-regulate. Multiple alternatively spliced variants, encoding the same protein, have been identified.

As used herein, the term “iRNA,” “RNAi”, “iRNA agent,” or “RNAi agent” refers to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript, e.g., via an RNA-induced silencing complex (RISC) pathway. In one embodiment, an iRNA as described herein effects inhibition of ALAS1 expression. Inhibition of ALAS1 expression may be assessed based on a reduction in the level of ALAS1 mRNA or a reduction in the level of the ALAS1 protein. As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an ALAS1 gene, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion. For example, the target sequence will generally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all sub-ranges therebetween. As non-limiting examples, the target sequence can be from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but are not limited to, G:U Wobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding an ALAS1 protein). For example, a polynucleotide is complementary to at least a part of an ALAS1 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding ALAS1. As another example, a polynucleotide is complementary to at least a part of an ALAS1 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding ALAS1.

The term “double-stranded RNA” or “dsRNA,” as used herein, refers to an iRNA that includes an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti-parallel and substantially complementary nucleic acid strands, which will be referred to as having “sense” and “antisense” orientations with respect to a target RNA. The duplex region can be of any length that permits specific degradation of a desired target RNA, e.g., through a RISC pathway, but will typically range from 9 to 36 base pairs in length, e.g., 15-30 base pairs in length. Considering a duplex between 9 and 36 base pairs, the duplex can be any length in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and any sub-range therein between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. dsRNAs generated in the cell by processing with Dicer and similar enzymes are generally in the range of 19-22 base pairs in length. One strand of the duplex region of a dsDNA comprises a sequence that is substantially complementary to a region of a target RNA. The two strands forming the duplex structure can be from a single RNA molecule having at least one self-complementary region, or can be formed from two or more separate RNA molecules. Where the duplex region is formed from two strands of a single molecule, the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a “hairpin loop”) between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucleotide; in some embodiments the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than a hairpin loop, the connecting structure is referred to as a “linker.” The term “siRNA” is also used herein to refer to a dsRNA as described above.

In another embodiment, the iRNA agent may be a “single-stranded siRNA” that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150: 883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein (e.g., sequences provided in Tables 2, 3, 6, 7, 8, 9, 14, and 15) may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

In another aspect, the RNA agent is a “single-stranded antisense RNA molecule”. An single-stranded antisense RNA molecule is complementary to a sequence within the target mRNA. Single-stranded antisense RNA molecules can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. Alternatively, the single-stranded antisense molecules inhibit a target mRNA by hybridizing to the target and cleaving the target through an RNaseH cleavage event. The single-stranded antisense RNA molecule may be about 10 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence. For example, the single-stranded antisense RNA molecule may comprise a sequence that is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense nucleotide sequences described herein, e.g., sequences provided in any one of Tables 2, 3, 6, 7, 8, 9, 14, and 15.

The skilled artisan will recognize that the term “RNA molecule” or “ribonucleic acid molecule” encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, a “ribonucleoside” includes a nucleoside base and a ribose sugar, and a “ribonucleotide” is a ribonucleoside with one, two or three phosphate moieties. However, the terms “ribonucleoside” and “ribonucleotide” can be considered to be equivalent as used herein. The RNA can be modified in the nucleobase structure or in the ribose-phosphate backbone structure, e.g., as described herein below. However, the molecules comprising ribonucleoside analogs or derivatives must retain the ability to form a duplex. As non-limiting examples, an RNA molecule can also include at least one modified ribonucleoside including but not limited to a 2′-O-methyl modified nucleostide, a nucleoside comprising a 5′ phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2′-deoxy-2′-fluoro modified nucleoside, a 2′-amino-modified nucleoside, 2′-alkyl-modified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof. Alternatively, an RNA molecule can comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the dsRNA molecule. The modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molecule. In one embodiment, modified RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA, e.g., via a RISC pathway.

In one aspect, a modified ribonucleoside includes a deoxyribonucleoside. In such an instance, an iRNA agent can comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang(s), or one or more deoxynucleosides within the double stranded portion of a dsRNA. However, it is self evident that under no circumstances is a double stranded DNA molecule encompassed by the term “iRNA.”

In one aspect, an RNA interference agent includes a single stranded RNA that interacts with a target RNA sequence to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleaves the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the invention relates to a single stranded RNA that promotes the formation of a RISC complex to effect silencing of the target gene.

As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) may be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′ end, 3′ end or both ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/or the 5′ end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/or the 5′ end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double-stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA or a plasmid from which an iRNA is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and in International Application No. WO 2009082817. These applications are incorporated herein by reference in their entirety.

“Introducing into a cell,” when referring to an iRNA, means facilitating or effecting uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; an iRNA may also be “introduced into a cell,” wherein the cell is part of a living organism. In such an instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be by a β-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, which are hereby incorporated by reference in their entirety. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or known in the art.

As used herein, the term “modulate the expression of,” refers to at an least partial “inhibition” or partial “activation” of an ALAS1 gene expression in a cell treated with an iRNA composition as described herein compared to the expression of ALAS1 in a control cell. A control cell includes an untreated cell, or a cell treated with a non-targeting control iRNA.

The terms “activate,” “enhance,” “up-regulate the expression of,” “increase the expression of,” and the like, in so far as they refer to an ALAS1 gene, herein refer to the at least partial activation of the expression of an ALAS1 gene, as manifested by an increase in the amount of ALAS1 mRNA, which may be isolated from or detected in a first cell or group of cells in which an ALAS1 gene is transcribed and which has or have been treated such that the expression of an ALAS1 gene is increased, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).

In one embodiment, expression of an ALAS1 gene is activated by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA as described herein. In some embodiments, an ALAS1 gene is activated by at least about 60%, 70%, or 80% by administration of an iRNA featured in the invention. In some embodiments, expression of an ALAS1 gene is activated by at least about 85%, 90%, or 95% or more by administration of an iRNA as described herein. In some embodiments, the ALAS1 gene expression is increased by at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000 fold or more in cells treated with an iRNA as described herein compared to the expression in an untreated cell. Activation of expression by small dsRNAs is described, for example, in Li et al., 2006 Proc. Natl. Acad. Sci. U.S.A. 103:17337-42, and in US20070111963 and US2005226848, each of which is incorporated herein by reference.

The terms “silence,” “inhibit expression of,” “down-regulate expression of,” “suppress expression of,” and the like, in so far as they refer to an ALAS1 gene, herein refer to the at least partial suppression of the expression of an ALAS1 gene, as assessed, e.g., based on on ALAS1 mRNA expression, ALAS1 protein expression, or another parameter functionally linked to ALAS1 gene expression (e.g., ALA or PBG concentrations in plasma or urine). For example, inhibition of ALAS1 expression may be manifested by a reduction of the amount of ALAS1 mRNA which may be isolated from or detected in a first cell or group of cells in which an ALAS1 gene is transcribed and which has or have been treated such that the expression of an ALAS1 gene is inhibited, as compared to a control. The control may be a second cell or group of cells substantially identical to the first cell or group of cells, except that the second cell or group of cells have not been so treated (control cells). The degree of inhibition is usually expressed as a percentage of a control level, e.g.,

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to ALAS1 gene expression, e.g., the amount of protein encoded by an ALAS1 gene, or the level of one or more porphyrins. The reduction of a parameter functionally linked to ALAS1 gene expression may similarly be expressed as a percentage of a control level. In principle, ALAS1 gene silencing may be determined in any cell expressing ALAS1, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given iRNA inhibits the expression of the ALAS1 gene by a certain degree and therefore is encompassed by the instant invention, the assays provided in the Examples below shall serve as such reference.

For example, in certain instances, expression of an ALAS1 gene is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA featured in the invention. In some embodiments, an ALAS1 gene is suppressed by at least about 60%, 65%, 70%, 75%, or 80% by administration of an iRNA featured in the invention. In some embodiments, an ALAS1 gene is suppressed by at least about 85%, 90%, 95%, 98%, 99%, or more by administration of an iRNA as described herein.

As used herein in the context of ALAS1 expression, the terms “treat,” “treating,” “treatment,” and the like, refer to relief from or alleviation of pathological processes related to ALAS1 expression (e.g., pathological processes involving porphyrins or defects in the porphyrin pathway, such as, for example, porphyrias). In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes related to ALAS1 expression), the terms “treat,” “treatment,” and the like mean to prevent, relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression or anticipated progression of such condition. For example, the methods featured herein, when employed to treat porphyria, may serve to reduce or prevent one or more symptoms associated with porphyria (e.g., pain), to reduce the severity or frequency of attacks associated with porphyria, to reduce the likelihood that an attack of one or more symptoms associated with porphyria will occur upon exposure to a precipitating condition, to shorten an attack associated with porphyria, and/or to reduce the risk of developing conditions associated with porphyria (e.g., hepatocellular cancer or neuropathy (e.g., progressive neuropathy),). Thus, unless the context clearly indicates otherwise, the terms “treat,” “treatment,” and the like are intended to encompass prophylaxis, e.g., prevention of disorders and/or symptoms of disorders related to ALAS1 expression.

By “lower” in the context of a disease marker or symptom is meant a statistically or clinically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is typically down to a level accepted as within the range of normal for an individual without such disorder.

As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes related to ALAS1 expression. The specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type of pathological process, the patient's history and age, the stage of pathological process, and the administration of other agents.

As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of an iRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an iRNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, in a method of treating a disorder related to ALAS1 expression (e.g., in a method of treating a porphyria), an effective amount includes an amount effective to reduce one or more symptoms associated with a porphyria, an amount effective to reduce the frequency of attacks, an amount effective to reduce the likelihood that an attack of one or more symptoms associated with porphyria will occur upon exposure to a precipitating factor, or an amount effective to reduce the risk of developing conditions associated with porphyria (e.g., neuropathy (e.g., progressive neuropathy), hepatocellular cancer). For example, if a given clinical treatment is considered effective when there is at least a 10% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 10% reduction in that parameter. For example, a therapeutically effective amount of an iRNA targeting ALAS1 can reduce ALAS1 protein levels by any measurable amount, e.g., by at least 10%, 20%, 30%, 40% or 50%.

The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. Agents included in drug formulations are described further herein below.

The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary from, for example, between 1% and 15% of the stated number or numerical range.

II. Double-Stranded Ribonucleic Acid (dsRNA)

Described herein are iRNA agents that inhibit the expression of an ALAS1 gene. In one embodiment, the iRNA agent includes double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of an ALAS1 gene in a cell or in a subject (e.g., in a mammal, e.g., in a human having a porphyria), where the dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of an ALAS1 gene, and where the region of complementarity is 30 nucleotides or less in length, generally 19-24 nucleotides in length, and where the dsRNA, upon contact with a cell expressing the ALAS1 gene, inhibits the expression of the ALAS1 gene by at least 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by Western blot. In one embodiment, the iRNA agent activates the expression of an ALAS1 gene in a cell or mammal. Expression of an ALAS1 gene in cell culture, such as in COS cells, HeLa cells, primary hepatocytes, HepG2 cells, primary cultured cells or in a biological sample from a subject can be assayed by measuring ALAS1 mRNA levels, such as by bDNA or TaqMan assay, or by measuring protein levels, such as by immunofluorescence analysis, using, for example, Western Blotting or flow cytometric techniques.

A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of an ALAS1 gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucleotides in length, inclusive. In some embodiments, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsRNAs having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, e.g., 15-30 nucleotides in length.

One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of 9 to 36, e.g., 15-30 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex of e.g., 15-30 base pairs that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, then, an miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target ALAS1 expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA as described herein may further include one or more single-stranded nucleotide overhangs. The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. In one embodiment, an ALAS1 gene is a human ALAS1 gene. In another embodiment the ALAS1 gene is a mouse or a rat ALAS1 gene. In specific embodiments, the first sequence is a sense strand of a dsRNA that includes a sense sequence from Table 2 or Table 3, and the second sequence is an antisense strand of a dsRNA that includes an antisense sequence from Table 2 or Table 3. In embodiments, the first sequence is a sense strand of a dsRNA that includes a sense sequence from Table 2, 3, 6, 7, 8, 9, 14, or 15, and the second sequence is an antisense strand of a dsRNA that includes an antisense sequence from Table 2, 3, 6, 7, 8, 9, 14, or 15. Alternative dsRNA agents that target sequences other than those of the dsRNAs of Table 2 or Table 3 can readily be determined using the target sequence and the flanking ALAS1 sequence.

In one aspect, a dsRNA will include at least sense and antisense nucleotide sequences, whereby the sense strand is selected from the groups of sequences provided in Tables 2 and 3, and the corresponding antisense strand of the sense strand is selected from Tables 2 and 3. In a further aspect, a dsRNA will include at least sense and antisense nucleotide sequences, whereby the sense strand is selected from the groups of sequences provided in Tables 2, 3, 6, 7, 8, 9, 14, and 15, and the corresponding antisense strand of the sense strand is selected from Tables 2, 3, 6, 7, 8, 9, 14, and 15. In these aspects, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated by the expression of an ALAS1 gene gene. As such, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in Table 2, 3, 6, 7, 8, 9, 14, or 15, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand from 2, 3, 6, 7, 8, 9, 14, or 15. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

The skilled person is well aware that dsRNAs having a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 2, 3, 6, 7, 8, 9, 14, and 15, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes having one of the sequences of Table 2, 3, 6, 7, 8, 9, 14, or 15 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Table 2, 3, 6, 7, 8, 9, 14, or 15, and differing in their ability to inhibit the expression of an ALAS1 gene by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated according to the invention.

In addition, the RNAs provided in Tables 2 and 3, as well as the RNAs provided in Tables 2, 3, 6, 7, 8, 9, 14, and 15, identify a site in an ALAS1 transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of such sequences. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least 15 contiguous nucleotides from one of the sequences provided in Tables 2, 3, 6, 7, 8, 9, 14, and 15 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in an ALAS1 gene.

While a target sequence is generally 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that may serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, in Tables 2, 3, 6, 7, 8, 9, 14, and 15, represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., in Tables 2, 3, 6, 7, 8, 9, 14, and 15, further optimization can be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those and sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) as an expression inhibitor.

An iRNA as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide iRNA agent RNA strand which is complementary to a region of an ALAS1 gene, the RNA strand generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of an ALAS1 gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of an ALAS1 gene is important, especially if the particular region of complementarity in an ALAS1 gene is known to have polymorphic sequence variation within the population.

In one embodiment, at least one end of a dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. In yet another embodiment, the RNA of an iRNA, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5′ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3′ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2′ position or 4′ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in this invention include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the modified RNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, each of which is herein incorporated by reference.

Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.

In other RNA mimetics suitable or contemplated for use in iRNAs, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

An iRNA may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.

The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

Representative U.S. Patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is herein incorporated by reference in its entirety.

Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861.

iRNA Motifs

In one embodiment, the sense strand sequence may be represented by formula (I):

(I) 5′ n_(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n_(q) 3′

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably YYY is all 2′-F modified nucleotides.

In one embodiment, the N_(a) and/or N_(b) comprise modifications of alternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand.

For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12 or 11, 12, 13) of − the sense strand, the count starting from the 1^(st) nucleotide, from the 5′-end; or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas:

(Ib) 5′ n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′; (Ic) 5′ n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q) 3′; or (Id) 5′ n_(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′.

When the sense strand is represented by formula (Ib), N_(b) represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a) independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ic), N_(b) represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a) can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Id), each N_(b) independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4, 5 or 6. Each N_(a) can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may be represented by the formula:

(Ia) 5′ n_(p)-N_(a)-YYY-N_(a)-n_(q) 3′.

When the sense strand is represented by formula (Ia), each N_(a) independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may be represented by formula (II):

(II) 5′ n_(q)′-N_(a)′-(Z′Z′Z′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(X′X′X′)_(l)-N′_(a)- n_(p)′ 3′

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)‘ and Y’ do not have the same modification;

and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications of alternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotide in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the 1^(st) nucleotide, from the 5′-end; or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end.

Preferably, the Y′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

The antisense strand can therefore be represented by the following formulas:

(IIb) 5′ n_(q)′-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(a)′-n_(p)′ 3′; (IIc) 5′ n_(q)′-N_(a)′-Y′Y′Y′-N_(b)′-X′X′X′-n_(p)′ 3′; or (IId) 5′ n_(q)′-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(b)′-X′X′X′-N_(a)′- n_(p)′ 3′.

When the antisense strand is represented by formula (IIb), N_(b)′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4, 5 or 6.

In other embodiments, k is 0 and l is 0 and the antisense strand may be represented by the formula:

(Ia) 5′ n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′-n_(q)′ 3′.

When the antisense strand is represented as formula (IIa), each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a 2′-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may contain YYY motif occurring at 9, 10 and 11 positions of the strand when the duplex region is 21 nt, the count starting from the 1^(st) nucleotide from the 5′-end, or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end; and Y represents 2′-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2′-OMe modification or 2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the 1^(st) nucleotide from the 5′-end, or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end; and Y′ represents 2′-O-methyl modification. The antisense strand may additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modifications at the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ each independently represents a 2′-OMe modification or 2′-F modification.

The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with a antisense strand being represented by any one of formulas (IIa), (IIb), (IIc), and (IId), respectively.

Accordingly, the RNAi agents for use in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex represented by formula (III):

(III) sense: 5′ n_(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(i)-N_(a)-n_(q) 3′ antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(i)-N_(a)′- n_(q)′ 5′

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

wherein

each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or may not be present, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and 1 are 0; or both k and 1 are 1.

Exemplary combinations of the sense strand and antisense strand forming a RNAi duplex include the formulas below:

(IIIa) 5′ n_(p)-N_(a)-YYY-N_(a)-n_(q) 3′ 3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′n_(q)′ 5′ (IIIb) 5′ n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′ 3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)n_(q) 5′ (IIIc) 5′ n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q) 3′ 3′ n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(a)′-n_(q)′ 5′ (IIId) 5′ n_(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′ 3′ n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)-n_(q)′ 5′

When the RNAi agent is represented by formula (IIIa), each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIb), each N_(b) independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIIc), each N_(b), N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIId), each N_(b), N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a), N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b) and N_(b)′ independently comprises modifications of alternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same or different from each other.

When the RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIc), and (IId), at least one of the Y nucleotides may form a base pair with one of the Y′ nucleotides. Alternatively, at least two of the Y nucleotides form base pairs with the corresponding Y′ nucleotides; or all three of the Y nucleotides all form base pairs with the corresponding Y′ nucleotides.

When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides may form a base pair with one of the Z′ nucleotides. Alternatively, at least two of the Z nucleotides form base pairs with the corresponding Z′ nucleotides; or all three of the Z nucleotides all form base pairs with the corresponding Z′ nucleotides.

When the RNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides may form a base pair with one of the X′ nucleotides. Alternatively, at least two of the X nucleotides form base pairs with the corresponding X′ nucleotides; or all three of the X nucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is different than the modification on the Y′ nucleotide, the modification on the Z nucleotide is different than the modification on the Z′ nucleotide, and/or the modification on the X nucleotide is different than the modification on the X′ nucleotide.

In one embodiment, when the RNAi agent is represented by formula (IId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. In another embodiment, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications and n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide a via phosphorothioate linkage. In yet another embodiment, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker. In another embodiment, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (IIIa), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In one embodiment, the RNAi agent is a multimer containing three, four, five, six or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, and one or both of the 3′ ends and are optionally conjugated to to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target same gene at two different target sites.

iRNA Conjugates

The iRNA agents disclosed herein can be in the form of conjugates. The conjugate may be attached at any suitable location in the iRNA molecule, e.g., at the 3′ end or the 5′ end of the sense or the antisense strand. The conjugates are optionally attached via a linker.

In some embodiments, an iRNA agent described herein is chemically linked to one or more ligands, moieties or conjugates, which may confer functionality, e.g., by affecting (e.g., enhancing) the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In some embodiments, a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Typical ligands will not take part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an a helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.

In some embodiments, the ligand is a GalNAc ligand that comprises one or more N-acetylgalactosamine (GalNAc) derivatives. Additional description of GalNAc ligands is provided in the section titled Carbohydrate Conjugates.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

Lipid Conjugates

In one embodiment, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule can typically bind a serum protein, such as human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control (e.g., inhibit) the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In one embodiment, the lipid based ligand binds HSA. For example, the ligand can bind HSA with a sufficient affinity such that distribution of the conjugate to a non-kidney tissue is enhanced. However, the affinity is typically not so strong that the HSA-ligand binding cannot be reversed.

In another embodiment, the lipid based ligand binds HSA weakly or not at all, such that distribution of the conjugate to the kidney is enhanced. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells.

Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).

Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, such as a helical cell-permeation agent. In one embodiment, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is typically an α-helical agent, and can have a lipophilic and a lipophobic phase. The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:3367). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:3368)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:3369)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 3370)) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Typically, the peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.

An RGD peptide moiety can be used to target a particular cell type, e.g., a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an dsRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver a iRNA agent to a tumor cell expressing α_(v)β₃ (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, an iRNA oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In one embodiment, a carbohydrate conjugate comprises a monosaccharide. In one embodiment, the monosaccharide is an N-acetylgalactosamine (GalNAc). GalNAc conjugates are described, for example, in U.S. Pat. No. 8,106,022, the entire content of which is hereby incorporated herein by reference. In some embodiments, the GalNAc conjugate serves as a ligand that targets the iRNA to particular cells. In some embodiments, the GalNAc conjugate targets the iRNA to liver cells, e.g., by serving as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).

In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives may be attached via a linker, e.g., a bivalent or trivalent branched linker. In some embodiments the GalNAc conjugate is conjugated to the 3′ end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3′ end of the sense strand) via a linker, e.g., a linker as described herein.

In some embodiments, the GalNAc conjugate is

In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic, wherein X is O or S

In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 1 and shown below

In some embodiments, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:

Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

(Formula XXIII), when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.

In one embodiment, an iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

Linkers

In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

In one embodiment, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXI)-(XXXIV):

wherein: q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different; P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C), T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C) are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O; Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C), are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O); R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C) are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) and L^(5C) represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXV):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

Ester-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

Peptide-Based Cleavable Linking Groups

In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)— (SEQ ID NO: 13), where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which is herein incorporated by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds, or “chimeras,” in the context of the present invention, are iRNA compounds, e.g., dsRNAs, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et a., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

Delivery of iRNA

The delivery of an iRNA to a subject in need thereof can be achieved in a number of different ways. In vivo delivery can be performed directly by administering a composition comprising an iRNA, e.g. a dsRNA, to a subject. Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.

Direct Delivery

In general, any method of delivering a nucleic acid molecule can be adapted for use with an iRNA (see e.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). However, there are three factors that are important to consider in order to successfully deliver an iRNA molecule in vivo: (a) biological stability of the delivered molecule, (2) preventing non-specific effects, and (3) accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example by direct injection or implantation into a tissue (as a non-limiting example, a tumor) or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that may otherwise be harmed by the agent or that may degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J., et al (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J., et al (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo.

Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to other groups, e.g., a lipid or carbohydrate group as described herein. Such conjugates can be used to target iRNA to particular cells, e.g., liver cells, e.g., hepatocytes. For example, GalNAc conjugates or lipid (e.g., LNP) formulations can be used to target iRNA to particular cells, e.g., liver cells, e.g., hepatocytes.

Lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O., et al (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H., et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al (2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

Vector Encoded iRNAs

In another aspect, iRNA targeting the ALAS1 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

An iRNA expression vector is typically a DNA plasmid or viral vector. An expression vector compatible with eukaryotic cells, e.g., with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors contain convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

An iRNA expression plasmid can be transfected into a target cell as a complex with a cationic lipid carrier (e.g., Oligofectamine) or a non-cationic lipid-based carrier (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance. Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct may be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.

Vectors useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.

Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgene.

In a specific embodiment, viral vectors that contain nucleic acid sequences encoding an iRNA can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding an iRNA are cloned into one or more vectors, which facilitates delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.

Adenoviruses are also contemplated for use in delivery of iRNAs. Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing an iRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

Use of Adeno-associated virus (AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.

Another typical viral vector is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

III. Pharmaceutical Compositions Containing iRNA

In one embodiment, the invention provides pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the iRNA is useful for treating a disease or disorder related to the expression or activity of an ALAS1 gene (e.g., a disorder involving the porphyrin pathway). Such pharmaceutical compositions are formulated based on the mode of delivery. For example, compositions can be formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery. In some embodiments, a composition provided herein (e.g., an LNP formulation) is formulated for intravenous delivery. In some embodiments, a composition provided herein (e.g., a composition comprising a GalNAc conjugate) is formulated for subcutaneous delivery.

The pharmaceutical compositions featured herein are administered in a dosage sufficient to inhibit expression of an ALAS1 gene. In general, a suitable dose of iRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose. The pharmaceutical composition may be administered once daily, or the iRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as can be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

The effect of a single dose on ALAS1 levels can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals.

The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.

Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes related to ALAS1 expression (e.g., pathological processes involving porphyrins or defects in the porphyrin pathway, such as, for example, porphyrias). Such models can be used for in vivo testing of iRNA, as well as for determining a therapeutically effective dose and/or an effective dosing regimen.

A suitable mouse model is, for example, a mouse containing a transgene expressing human ALAS1. Mice that have knock-in mutations (e.g., mutations that are associated with acute hepatic porphyrias in humans) can be used to determine the therapeutically effective dosage and/or duration of administration of ALAS1 siRNA. The present invention also includes pharmaceutical compositions and formulations that include the iRNA compounds featured in the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.

The iRNA can be delivered in a manner to target a particular tissue, such as a tissue that produces erythrocytes. For example, the iRNA can be delivered to bone marrow, liver (e.g., hepatocyes of liver), lymph glands, spleen, lungs (e.g., pleura of lungs) or spine. In one embodiment, the iRNA is delivered to bone marrow.

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

Liposomal Formulations

There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

In order to traverse intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex.

The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M)1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C_(215G), that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

Nucleic Acid Lipid Particles

In one embodiment, an ALAS1 dsRNA featured in the invention is fully encapsulated in the lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G), or a mixture thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or a PEG-distearyloxypropyl (C]₈). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

In some embodiments, the iRNA is formulated in a lipid nanoparticle (LNP).

LNP01

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is herein incorporated by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (e.g., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid-dsRNA formulations are provided in the following table.

TABLE 10 Examplary lipid formulations cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugate Cationic Lipid Lipid:siRNA ratio SNALP 1,2-Dilinolenyloxy-N,N- DLinDMA/DPPC/Cholesterol/PEG-cDMA dimethylaminopropane (DLinDMA) (57.1/7.1/34.4/1.4) lipid: siRNA ~ 7:1 S-XTC 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DPPC/Cholesterol/PEG-cDMA [1,3]-dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid: siRNA ~ 7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxo1ane (XTC) 57.5/7.5/31.5/3.5 lipid: siRNA ~ 6:1 LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~ 11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid: siRNA ~ 6:1 LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~ 11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2- ALN100/DSPC/Cholesterol/PEG-DMG di((9Z,12Z)-octadeca-9,12- 50/10/38.5/1.5 dienyl)tetrahydro-3aH- Lipid: siRNA 10:1 cyclopenta[d][1,3]dioxo1-5-amine (ALN100) LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta- MC-3/DSPC/Cholesterol/PEG-DMG 6,9,28,31-tetraen-19-yl 4- 50/10/38.5/1.5 (dimethylamino)butanoate (MC3) Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2- C12-200/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2- 50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin- Lipid:siRNA 10:1 1-yl)ethylazanediyl)didodecan-2-ol (C12-200) LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3 MC3/DSPC/Chol/PEG-DSG/Ga1NAc- PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC: distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000) SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. W02009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference. XTC comprising formulations are described, e.g., in U.S. Provisional Serial No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Serial No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun. 10, 2009; U.S. Provisional Serial No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Serial No. 61/239,686, filed Sep. 3, 2009, and International Application No. PCT/U52010/022614, filed Jan. 29, 2010, which are hereby incorporated by reference. MC3 comprising formulations are described, e.g., in U.S. Provisional Serial No. 61/244,834, filed Sep. 22, 2009, U.S. Provisional Serial No. 61/185,800, filed Jun. 10, 2009, and International Application No. PCT/US10/28224, filed Jun. 10, 2010, which are hereby incorporated by reference. ALNY-100 comprising formulations are described, e.g., International patent application number PCT/U509/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference. C12-200 comprising formulations are described in U.S. Provisional Serial No. 61/175,770, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.

Synthesis of Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles featured in the invention may be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless indicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.

Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.

“Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acyl groups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted heterocycle” means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, —CN, —OR^(x), —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y), wherein n is 0, 1 or 2, R^(x) and R^(y) are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —OR^(x), heterocycle, —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R, —C(═O)OR, —C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y).

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods featured in the invention may require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an “alcohol protecting group” is used. An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

Synthesis of Formula A

In one embodiments, nucleic acid-lipid particles featured in the invention are formulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, the lipid of formula A above may be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.

Lipid A, where R₁ and R₂ are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R₃ and R₄ are independently lower alkyl or R₃ and R₄ can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed, yielding a colorless oil (0.54 g).

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:

Synthesis of 515:

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THE in a two neck RBF (1 L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0° C. and quenched with careful addition of saturated Na2SO4 solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

Synthesis of 516:

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1×100 mL) and saturated NaHCO₃ solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl₃, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H]−232.3 (96.94%).

Synthesis of 517A and 517B:

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (˜3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL) followed by saturated NaHCO₃(1×50 mL) solution, water (1×30 mL) and finally with brine (1×50 mL). Organic phase was dried over an.Na2SO4 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield: −6 g crude

517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS—[M+H]−266.3, [M+NH4+]−283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518:

Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl₃, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519:

A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40° C. over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR=130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc. 654.6, Found 654.6.

Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations featured in the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions featured in the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Additional Formulations

Emulsions

The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.

Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of β-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293fectin™ (Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison, Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent (Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^(a) D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVec™/LipoGen™ (Invivogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFect™ (B-Bridge International, Mountain View, Calif., USA), among others.

Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Other Components

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more biologic agents which function by a non-RNAi mechanism. Examples of such biologic agents include agents that interfere with an interaction of ALAS1 and at least one ALAS1 binding partner.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are typical.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in combination with other known agents effective in treatment of diseases or disorders related to ALAS1 expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

Methods for Treating Diseases Related to Expression of an ALAS1 Gene

The invention relates in particular to the use of an iRNA targeting ALAS1 to inhibit ALAS1 expression and/or to treat a disease, disorder, or pathological process that is related to ALAS1 expression.

As used herein, “a disorder related to ALAS1 expression,” a “disease related to ALAS1 expression, a “pathological process related to ALAS1 expression,” or the like includes any condition, disorder, or disease in which ALAS1 expression is altered (e.g., elevated), the level of one or more porphyrins is altered (e.g., elevated), the level or activity of one or more enzymes in the heme biosynthetic pathway (porphyrin pathway) is altered, or other mechanisms that lead to pathological changes in the heme biosynthetic pathway. For example, an iRNA targeting an ALAS1 gene, or a combination thereof, may be used for treatment of conditions in which levels of a porphyrin or a porphyrin precursor (e.g., ALA or PBG) are elevated (e.g., certain porphyrias), or conditions in which there are defects in the enzymes of the heme biosynthetic pathway (e.g., certain porphyrias). Disorders related to ALAS1 expression include, for example, X-linked sideroblastic anemia (XLSA), ALA deyhdratase deficiency porphyria (Doss porphyria), acute intermittent porphyria (AIP), congenital erythropoietic porphyria, prophyria cutanea tarda, hereditary coproporphyria (coproporphyria), variegate porphyria, erythropoietic protoporphyria (EPP), and transient erythroporphyria of infancy.

As used herein, a “subject” to be treated according to the methods described herein, includes a human or non-human animal, e.g., a mammal. The mammal may be, for example, a rodent (e.g., a rat or mouse) or a primate (e.g., a monkey). In some embodiments, the subject is a human.

In some embodiments, the subject is suffering from a disorder related to ALAS1 expression (e.g., has been diagnosed with a porphyria or has suffered from one or more symptoms of porphyria and is a carrier of a mutation associated with porphyria) or is at risk of developing a disorder related to ALAS1 expression (e.g., a subject with a family history of porphyria, or a subject who is a carrier of a genetic mutation associated with porphyria).

Classifications of porphyrias, including acute hepatic porphyrias, are described, e.g., in Balwani, M. & Desnick, R. J., Blood, 120(23), published online as Blood First Edition paper, July 12, 102; DOI 10.1182/blood-2012-05-423186. As described in Balwain & Desnick, acute intermittent porphyria (AIP) hereditary coproporphyria (HCP), variegate porphyria (VP) are autosomal dominant porphyrias and ALA deyhdratase deficiency porphyria (ADP) is autosomal recessive. In rare cases, AIP, HCP, and VP occur as homozygous dominant forms. In addition, there is a rare homozygous recessive form of porphyria cutanea tarda (PCT), which is the single hepatic cutaneous porphyria, and is also known as hepatoerythropoietic porphyria. The clinical and laboratory features of these porphyrias are described in Table 11 below.

TABLE 11 Human hepatic porphyrias: clinical and laboratory features Enzyme Principal activity, Deficient symptoms, % of Increased porphyrin precursors and/or porphyrins* Porphyria enzyme Inheritance NV or CP normal Erythrocytes Urine Stool Acute hepatic porphyrias ADP ALA- AR NV ~5 Zn-protoporphyrin ALA, — dehydratase coproporphyrin III AIP HMB- AD NV ~50 — ALA, PBG, — synthase uroporphyrin HCP COPRO- AD NV and CP ~50 — ALA, PBG, coproporphyrin oxidase coproporphyrin III III VP PROTO- AD NV and CP ~50 — ALA, PBG coproporphyrin oxidase coproporphyrin III, III protoporphyrin Hepatic cutaneous porphyrias PCT URO- Sporadic or CP <20 — uroporphyrin, uroporphyrin, 7- decarboxylase AD 7-carboxylate carboxylate porphyrin porphyrin AR indicates autosomal recessive; AD, autosomal dominant; NV, neurovisceral; CP, cutaneous photosensitivity; and —, not applicable. *Increases that may be important for diagnosis.

In some embodiments, the subject has or is at risk for developing a porphyria, e.g., a hepatic porphyria, e.g., AIP, HCP, VP, ADP, or hepatoerythropoietic porphyria.

In some embodiments, the porphyria is an acute hepatic porphyria, e.g., an acute hepatic porphyria is selected from acute intermittent porphyria (AIP), hereditary coproporphyria (HCP), variegate porphyria (VP), and ALA deyhdratase deficiency porphyria (ADP).

In some embodiments, the porphyria is a dual porphyria, e.g., at least two porphyrias. In some embodiments, the dual porphyria comprises two or more porphyrias selected from acute intermittent porphyria (AIP) hereditary coproporphyria (HCP), variegate porphyria (VP), and ALA deyhdratase deficiency porphyria (ADP).

In some embodiments, the porphyria is a homozygous dominant hepatic porphyria (e.g., homozygous dominant AIP, HCP, or VP) or hepatoerythropoietic porphyria, In some embodiments, the porphyria is AIP, HCP, VP, or hepatoerythropoietic porphyria, or a combination thereof (e.g., a dual porphyria). In embodiments, the AIP, HCP, or VP is either heterozygous dominant or homozygous dominant.

In embodiments, the subject has or is at risk for developing a porphyria, e.g., ADP, and shows an elevated level (e.g., an elevated urine level) of ALA and/or coproporphyrin III. In embodiments, the subject has or is at risk for developing a porphyria, e.g., ADP, and shows an elevated level of erythrocyte Zn-protoporphyrin.

In embodiments, the subject has or is at risk for developing a porphyria, e.g., AP, and shows an elevated level (e.g., an elevated urine level) of ALA, PBG, and/or uroporphyrin.

In embodiments, the subject has or is at risk for developing a porphyria, e.g., HCP, and shows an elevated level (e.g., an elevated urine level) of ALA, PBG, and/or coproporphyrin III. In embodiments, the subject has or is at risk for developing a porphyria, e.g., HCP, and shows an elevated level (e.g., an elevated stool level) of coproporphyrin III.

In embodiments, the subject has or is at risk for developing a porphyria, e.g., VP, and shows an elevated level (e.g., an elevated urine level) of ALA, PBG, and/or coproporphyrin III.

In embodiments, the subject has or is at risk for developing a porphyria, e.g., HCP, and shows an elevated level (e.g., an elevated stool level) of coproporphyrin III and/or protoporphyrin.

In embodiments, the subject has or is at risk for developing a porphyria, e.g., PCT, (e.g., hepatoerythropoietic porphyria) and shows an elevated level (e.g., an elevated urine level) of uroporphyrin and/or 7-carboxylate porphyrin. In embodiments, the subject has or is at risk for developing a porphyria, e.g., PCT, (e.g., hepatoerythropoietic porphyria) and shows an elevated level (e.g., an elevated stool level) of uroporphyrin and/or 7-carboxylate porphyrin.

A mutation associated with porphyria includes any mutation in a gene encoding an enzyme in the heme biosynthetic pathway (porphyrin pathway) or a gene which alters the expression of a gene in the heme biosynthetic pathway. In many embodiments, the subject carries one or more mutations in an enzyme of the porphyrin pathway (e.g., a mutation in ALA dehydratase or PBG deaminase). In some embodiments, the subject is suffering from an acute porphyria (e.g., AIP, ALA deydratase deficiency porphyria).

In some cases, patients with an acute hepatic porphyria (e.g., AIP), or patients who carry mutations associated with an acute hepatic porphyria (e.g., AIP) but who are asymptomatic, have elevated ALA and/or PBG levels compared with healthy individuals. See, e.g., Floderus, Y. et al, Clinical Chemistry, 52(4): 701-707, 2006; Sardh et al., Clinical Pharmacokinetics, 46(4): 335-349, 2007. In such cases, the level of ALA and/or PBG can be elevated even when the patient is not having, or has never had, an attack. In some such cases, the patient is otherwise completely asymptomatic. In some such cases, the patient suffers from pain, e.g., neuropathic pain, which can be chronic pain (e.g., chronic neuropathic pain). In some cases, the patient has a neuropathy. In some cases, the patient has a progressive neuropathy.

In some embodiments, the subject to be treated according to the methods described herein has an elevated level of a porphyrin or a porphyrin precursor, e.g., ALA and/or PBG. Levels of a porphyrin or a porphyrin precursor can be assessed using methods known in the art or methods described herein. For example, methods of assessing using and plasma ALA and PBG levels, as well as urine and plasma porphyrin levels, are disclosed in Floderus, Y. et al, Clinical Chemistry, 52(4): 701-707, 2006; and Sardh et al., Clinical Pharmacokinetics, 46(4): 335-349, 2007, the entire contents of which are hereby incorporated in their entirety.

In some embodiments, the subject is an animal model of a porphyria, e.g., a mouse model of a porphyria (e.g., a mutant mouse as described in Lindberg et al. Nature Genetics, 12: 195-199, 1996). In some embodiments, the subject is a human, e.g., a human who has or is at risk for developing a porphyria, as described herein. In some embodiments, the subject is not having an acute attack of porphyria. In some embodiments, the subject has never had an attack. In some embodiments, the patient suffers from chronic pain. In some embodiments, the patient has nerve damage. In embodiments, the subject has EMG changes and/or changes in nerve conduction velocity. In some embodiments, the subject is asymptomatic. In some embodiments, the subject is at risk for developing a porphyria (e.g., carries a gene mutation associated with a porphyria) and is asymptomatic. In some embodiments, the subject has previously had an acute attack but is asymptomatic at the time of treatment.

In some embodiments, the subject is at risk for developing a porphyria and is treated prophylactically to prevent the development of a porphyria. In some embodiments the subject has an elevated level of a porphyrin or a porphyrin precursor, e.g., ALA and/or PBG. In some embodiments, the prophylactic treatment begins at puberty. In some embodiments the treatment lowers the level (e.g., the plasma level or the urine level) of a porphyrin or a porphyrin precursor, e.g., ALA and/or PBG. In some embodiments, the treatment prevents the development of an elevated level of a porphyrin or a porphyrin precursor, e.g., ALA and/or PBG. In some embodiments, the treatment prevents the development of, or decreases the frequency or severity of, a symptom associated with a porphyria, e.g., pain or nerve damage.

In some embodiments, the level of a porphyrin or a porphyrin precursor, e.g., ALA or PBG, is elevated, e.g., in a sample of plasma or urine from the subject. In some embodiments, the level of a porphyrin or a porphyrin precursor, e.g., ALA or PBG, in the subject is assessed based on the absolute level of the porphyrin or the porphyrin precursor, e.g., ALA or PBG in a sample from the subject. In some embodiments, the level of a porphyrin or a porphyrin precursor, e.g., ALA or PBG, in the subject is assessed based on the relative level of the porphyrin or porphyrin precursor, e.g., ALA or PBG, in a sample from the subject. In some embodiments, the relative level is relative to the level of another protein or compound, e.g., the level of creatinine, in a sample from the subject. In some embodiments, the sample is a urine sample. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a stool sample.

An elevated level of a porphyrin or a porphyrin precursor, e.g., ALA and/or PBG, can be established, e.g., by showing that the subject has a level of a porphyrin or a porphyrin precursor, e.g., ALA and/or PBG (e.g., a plasma or urine level of ALA and/or PBG) that is greater than, or greater than or equal to, a reference value. A physician with expertise in the treatment of porphyrias would be able to determine whether the level of a porphyrin or a porphyrin precursor, (e.g., ALA and/or PBG) is elevated, e.g., for the purpose of diagnosing a porphyria or for determining whether a subject is at risk for developing a porphyria, e.g., a subject may be predisposed to an acute attack or to pathology associated with a porphyria, such as, e.g., chronic pain (e.g., neuropathic pain) and neuropathy (e.g., progressive neuropathy).

As used herein, a “reference value” refers to a value from the subject when the subject is not in a disease state, or a value from a normal or healthy subject, or a value from a reference sample or population, e.g., a group of normal or healthy subjects (e.g., a group of subjects that does not carry a mutation associated with a porphyria and/or a group of subjects that does not suffer from symptoms associated with a porphyria).

In some embodiments, the reference value is a pre-disease level in the same individual. In some embodiments, the reference value is a level in a reference sample or population. In some embodiments, the reference value is the mean or median value in a reference sample or population. In some embodiments, the reference value the value that is is two standard deviations above the mean in a reference sample or population. In some embodiments, the reference value is the value that is 2.5, 3, 3.5, 4, 4.5, or 5 standard deviations above the mean in a reference sample or population.

In some embodiments, wherein the subject has an elevated level of a porphyrin or a porphyrin precursor, e.g., ALA and/or PBG, the subject has a level of ALA and/or PBG that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than a reference value. In some embodiments, the subject has a level of a porphyrin or a porphyrin precursor, e.g., ALA and/or PBG, that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold higher than a reference value.

In some embodiments, the reference value is an upper reference limit. As used herein, an “upper reference limit” refers to a level that is the upper limit of the 95% confidence interval for a reference sample or population, e.g., a group of normal (e.g., wild type) or healthy individuals, e.g., individuals who do not carry a genetic mutation associated with a porphyria and/or individuals who do not suffer from a porphyria. Accordingly, a lower reference limit refers to a level that is the lower limit of the same 95% confidence interval.

In some embodiments wherein the subject has an elevated level, e.g., a plasma level or a urine level, of a porphyrin or a porphyrin precursor, e.g., ALA or PBG, the level is greater than or equal to 2 times, 3 times, 4 times, or 5 times that of a reference value, e.g., an upper reference limit. In some embodiments, the subject has a urine level of a porphyrin or a porphyrin precursor, e.g., ALA or PBG, that is greater than 4 times that of an upper reference limit.

In some embodiments, the reference value is a value provided in Floderus, Y. et al, Clinical Chemistry, 52(4): 701-707, 2006 or Sardh et al., Clinical Pharmacokinetics, 46(4): 335-349, 2007. In some embodiments, the reference value is a value provided in Table 1 of Sardh et al.

In some embodiments, the subject is a human and has a urine level of PBG that is greater than or equal to 4.8 mmol/mol creatinine. In certain embodiments, the subject is a human and has a urine level of PBG that is greater than, or greater than or equal to, about 3, 4, 5, 6, 7, or 8 mmol/mol creatinine.

In embodiments, the reference value for plasma PBG is 0.12 μmol/L. In embodiments, the subject is a human and has a plasma PBG level that is greater than, or greater than or equal to, 0.10 μmol/L, 0.12 μmol/L, 0.24 μmol/L, 0.36 μmol/L, 0.48 μmol/L, or 0.60 μmol/L. In embodiments, the subject is a human and has a plasma level of PBG that is greater than, or greater than or equal to, 0.48 μmol/L.

In embodiments, the reference value for urine PBG is 1.2 mmol/mol creatinine. In embodiments, the subject is a human and has a urine PBG level that is greater than, or greater than or equal to, 1.0 mmol/mol creatinine, 1.2 mmol/mol creatinine, 2.4 mmol/mol creatinine, 3.6 mmol/mol creatinine, 4.8 mmol/mol creatinine, or 6.0 mmol/mol creatinine. In embodiments, the subject is a human and has a urine level of PBG that is greater than, or greater than or equal to, 4.8 mmol/mol creatinine.

In embodiments, the reference value for plasma ALA is 0.12 μmol/L. In embodiments, the subject is a human and has a plasma ALA level that is greater than, or greater than or equal to, 0.10 μmol/L, 0.12 μmol/L, 0.24 μmol/L, 0.36 μmol/L, 0.48 μmol/L, or 0.60 μmol/L. In embodiments, the subject is a human and has a plasma ALA level that is greater than, or greater than or equal to 0.48 μmol/L.

In embodiments, the reference value for urine ALA is 3.1 mmol/mol creatinine. In embodiments, the subject is a human and has a urine ALA level that is greater than, or greater than or equal to, 2.5 mmol/mol creatinine, 3.1 mmol/mol creatinine, 6.2 mmol/mol creatinine, 9.3 mmol/mol creatinine, 12.4 mmol/mol creatinine, or 15.5 mmol/mol creatinine.

In embodiments, the reference value for plasma porphyrin is 10 nmol/L. In embodiments, the subject is a human and has a plasma porphyrin level that is greater than, or greater than or equal to, 10 nmol/L. In embodiments, the subject is a human and has a plasma porphyrin level that is greater than, or greater than or equal to, 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nmol/L. the subject is a human and has a plasma porphyrin level that is greater than, or greater than or equal to 40 nmol/L. In embodiments, the reference value for urine porphyrin is 25 μmol/mol creatinine. In embodiments, the subject is a human and has a urine porphyrin level that is greater than, or greater than or equal to, 25 μmol/mol creatinine. In embodiments, the subject is a human and has a urine porphyrin level that is greater than, or equal to, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 μmol/mol creatinine.

In some embodiments, the subject has a level, e.g., a plasma level or a urine level, of a porphyrin or a porphyrin precursor, e.g., ALA or PBG, that is greater than that of 99% of individuals in a sample of healthy individuals.

In some embodiments, the subject has a level, e.g., a plasma level or a urine level, of ALA or PBG that is greater than two standard deviations above the mean level in a sample of healthy individuals.

In some embodiments, the subject has a urine level of ALA that is 1.6 or more times that of the mean level in a normal subject (e.g., a subject that does not carry a mutation associated with a porphyria). In some embodiments, the subject has a plasma level of ALA that is 2 or 3 times that of the mean level in a normal subject. In some embodiments, the subject has a urine level of PBG that is four or more times that of the mean level in a normal subject. In some embodiments, the subject has a plasma level of PBG that is four or more times that of the mean level in a normal subject.

In some embodiments, the method is effective to decrease the level of a porphyrin or a porphyrin precursor, e.g., ALA and/or PBG. In embodiments, the method is effective to produce a predetermined reduction in the elevated level of the porphyrin or porphyrin precursor, e.g., ALA or PBG. In some embodiments, the predetermined reduction is a decrease of at least 10%, 20%, 30%, 40%, or 50%. In some embodiments, the predetermined reduction is a reduction that is effective to prevent or ameliorate symptoms, e.g., pain or recurring attacks.

In some embodiments, the predetermined reduction is a reduction that is at least 1, 2, 3, or more standard deviations, wherein the standard deviation is determined based on the values from a reference sample, e.g., a reference sample as described herein.

In some embodiments, the predetermined reduction is a reduction that brings the level of the porphyrin or porphyrin precursor to a level that is less than, or to a level that is less than or equal to, a reference value (e.g., a reference value as described herein).

In some embodiments, the subject to be treated according to the methods described suffers from pain, e.g., chronic pain. In some embodiments, the subject has or is at risk for developing a porphyria, e.g. an acute hepatic porphyria, e.g., AIP. In embodiments, the method is effective to treat the pain, e.g., by reducing the severity of the pain or curing the pain. In embodiments, the method is effective to decrease or prevent nerve damage.

In some embodiments, the subject to be treated according to the methods described herein (a) has an elevated level of ALA and/or PBG and (b) suffers from pain, e.g., chronic pain. In embodiments, the method is effective to decrease an elevated level of ALA and/or PBG and/or to treat the pain, e.g., by reducing the severity of the pain or curing the pain.

In some embodiments, the subject is an animal that serves as a model for a disorder related to ALAS1 expression.

In some embodiments the subject is an animal that serves as a model for porphyria (e.g., a genetically modified animal with one or more mutations. In some embodiments, the porphyria is AIP and the subject is an animal model of AIP. In one such embodiment, the subject is a genetically modified mouse that is deficient in porphobilinogen deaminase, such as, for example, the mouse described in Lindberg et al., Nature Genetics, 12:195-199, 1996, or the homozygous R167Q mouse described in Yasuda, M., Yu, C. Zhang, J., Clavero, S., Edelmann, W., Gan, L., Phillips, J. D., & Desnick, R. J. Acute intermittent porphyria: A severely affected knock-in mouse that mimics the human homozygous dominant phenotype. (Abstract of Presentation on Oct. 14, 2011 at the American Society of Human Genetics; Program No. 1308F; accessed online on Apr. 4, 2012 at ichg2011.org/cgi-bin/showdetail.pl?absno=21167); both of these references are hereby incorporated herein in their entirety. Several knock-in models for mutations causing homozygous dominant AIP in humans have been generated. The mutations employed include, e.g., R167Q, R173Q, and R173W in PBG deaminase. Viable homozygotes included the R167Q/R176Q and R167Q/R173Q, both of which exhibit constitutively elevated ALA and PBG levels analogous to the phenotype in human homozygous dominant AIP; in some embodiments, such a viable homozygous ATP mouse model is the subject.

In one embodiment, a subject to be treated according to the methods described herein, (e.g., a human subject or patient), is at risk of developing, or has been diagnosed, with a disorder related to ALAS1 expression, e.g. a porphyria. In some embodiments, the subject is a subject who has suffered one or more acute attacks of one or more porphyric symptoms. In other embodiments, the subject is a subject who has suffered chronically from one or more symptoms of porphyria (e.g., pain, e.g., neuropathic pain and or neuropathy, e.g., progressive neuropathy). In some embodiments, the subject carries a genetic alteration (e.g., a mutation) as described herein but is otherwise asymptomatic. In some embodiments, the subject has previously been treated with a heme product (e.g., hemin, heme arginate, or heme albumin), as described herein.

In some embodiments, a subject (e.g., a subject with a porphyria, such as, e.g., AP) to be treated according to the methods described herein has recently experienced or is currently experiencing a prodrome. In some such embodiments, the subject is administered a combination treatment, e.g., an iRNA as described herein, and one or more additional treatments known to be effective against porphyria (e.g., glucose and/or a heme product such as hemin, as described herein) or its associated symptoms.

In one embodiment, an iRNA as described herein is administered in combination with glucose or dextrose. For example, 10-20% dextrose in normal saline may be provided intravenously. Typically, when glucose is administered, at least 300 g of 10% glucose is administered intravenously daily. The iRNA (e.g., an iRNA in an LNP formulation) may also be administered intravenously, as part of the same infusion that is used to administer the glucose or dextrose, or as a separate infusion that is administered before, concurrently, or after the administration of the glucose or dextrose. In some embodiments, the iRNA is administered via a different route of administration (e.g., subcutaneously). In yet another embodiment, the iRNA is administered in combination with total parenteral nutrition. The iRNA may be administered before, concurrent with, or after the administration of total parenteral nutrition.

In one embodiment, the iRNA is administered in combination with a heme product (e.g., hemin, heme arginate, or heme albumin). In a further embodiment, the iRNA is administered in combination with a heme product and glucose, a heme product and dextrose, or a heme product and total parenteral nutrition.

A “prodrome,” as used herein, includes any symptom that the individual subject has previously experienced immediately prior to developing an acute attack. Typical symptoms of a prodrome include, e.g., abdominal pain, nausea, headaches, psychological symptoms (e.g., anxiety), restlessness and/or insomnia. In some embodiments, the subject experiences pain (e.g., abdominal pain and/or a headache) during the prodrome. In some embodiments, the subject experiences nausea during the prodrome. In some embodiments, the subject experiences psychological symptoms (e.g., anxiety) during the prodrome. In some embodiments, the subject becomes restless and/or suffers from insomnia during the prodrome.

An acute “attack” of porphyria involves the onset of one or more symptoms of porphyria, typically in a patient who carries a mutation associated with porphyria (e.g., a mutation in a gene that encodes an enzyme in the porphyrin pathway).

In certain embodiments, administration of an ALAS1 iRNA results in a decrease in the level of one or more porphyrins or porphyrin precursors, as described herein (e.g., ALA and/or PBG). The decrease may be measured relative to any appropriate control or reference value. For example, the decrease in the level of one or more porphyrins or porphyrin precursors may be established in an individual subject, e.g., as a decrease of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more compared with the level prior to treatment (e.g., immediately prior to treatment). A decrease in the level of a porphyrin precursor, a porphyrin, or or a porphyrin metabolite may be measured using any method known in the art. For example, the level of PBG and/or ALA in urine or plasma may be assessed, using the Watson-Schwartz test, ion exchange chromatography, or high-performance liquid chromatography-mass spectrometry. See, e.g., Thunell (1993).

In some embodiments, administration of an ALAS1 siRNA is effective to reduce the level of ALA and/or PBG in the subject. The level of ALA or PBG in the subject can be assessed, e.g., based on the absolute level of ALA or PBG, or based on the relative level of ALA or PBG (e.g., relative to the level of another protein or compound, e.g., the level of creatinine) in a sample from the subject. In some embodiments, the sample is a urine sample. In some embodiments, the sample is a plasma sample.

In certain embodiments, an iRNA that targets ALAS1 is administered in combination one or more additional treatments, e.g., another treatment known to be effective in treating porphyria or symptoms of porphyria. For example, the other treatment may be glucose (e.g., IV glucose) or a heme product (e.g., hemin, heme arginate, or heme albumin). The additional treatment(s) may be administered before, after, or concurrent with the administration of iRNA.

The iRNA and an additional therapeutic agent can be administered in combination in the same composition, e.g., intravenously, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.

In some embodiments, administration of iRNA, or administration of iRNA in combination one or more additional treatments (e.g., glucose, dextrose or the like), decreases the frequency of acute attacks (e.g., by preventing acute attacks so that they no longer occur, or by reducing the number of attacks that occur in a certain time period, e.g., fewer attacks occur per year). In some such embodiments, the iRNA is administered according to a regular dosing regimen, e.g., daily, weekly, biweekly, or monthly.

In some embodiments, the iRNA is administered after an acute attack of porphyria. In some such embodiments, the iRNA is in a composition, e.g. a composition comprising a lipid formulation, e.g. an LNP formulation.

In some embodiments, the iRNA is administered during an acute attack of porphyria. In some such embodiments, the iRNA is in a composition, e.g. a composition comprising a lipid formulation (e.g., an LNP formulation) or a composition comprising a GalNAc conjugate.

In some embodiments, administration of an ALAS1 siRNA is effective to lessen the severity of the attack (e.g., by ameliorating one or more signs or symptoms associated with the attack). In some embodiments, administration of an ALAS1 siRNA is effective to shorten the duration of an attack. In some embodiments, administration of an ALAS1 siRNA is effective to stop an attack. In some embodiments, the iRNA is administered prophylactically to prevent an acute attack of porphyria. In some such embodiments, the iRNA is in the form of a GalNAc conjugate, e.g., in a composition comprising a GalNAc conjugate. In some embodiments, the prophylactic administration is before, during, or after exposure to or occurrence of a precipitating factor. In some embodiments, the subject is at risk of developing porphyria.

In some embodiments, the siRNA is administered during a prodrome. In some embodiments, the prodrome is characterized by pain (e.g., headache and/or abdominal pain), nausea, psychological symptoms (e.g., anxiety), restlessness and/or insomnia.

In some embodiments, the siRNA is administered during a particular phase of the menstrual cycle, e.g., during the luteal phase.

In some embodiments, administration of an ALAS1 siRNA is effective to prevent attacks (e.g., recurrent attacks that are associated with a prodrome and/or with a precipitating factor, e.g., with a particular phase of the menstrual cycle, e.g., the luteal phase). In some embodiments, administration of an ALAS1 siRNA is effective to reduce the frequency of attacks. In embodiments, administration of an ALAS1 siRNA is effective to lessen the severity of the attack (e.g., by ameliorating one or more signs or symptoms associated with the attack). In some embodiments, administration of an ALAS1 siRNA is effective to shorten the duration of an attack. In some embodiments, administration of an ALAS1 siRNA is effective to stop an attack.

In some embodiments administration of an ALAS1 siRNA is effective to prevent or decrease the frequency or severity of pain, e.g., neuropathic pain.

In some embodiments administration of an ALAS1 siRNA is effective to prevent or decrease the frequency or severity of neuropathy

Effects of administration of an ALAS1 siRNA can be established, for example, by comparison with an appropriate control. For example, a decrease in the frequency of acute attacks, as well as a decrease in the level of one or more porphyrins or porphyrin precursors, may be established, for example, in a group of patients with AIP, as a decreased frequency compared with an appropriate control group. A control group (e.g., a group of similar individuals or the same group of individuals in a crossover design) may include, for example, an untreated population, a population that has been treated with a conventional treatment for porphyria (e.g., a conventional treatment for AIP may include glucose, hemin, or both); a population that has been treated with placebo, or a non-targeting iRNA, optionally in combination with one or more conventional treatments for porphyria (e.g., glucose, e.g., IV glucose), and the like.

A subject “at risk” of developing porphyria, as used herein, includes a subject with a family history of porphyria and/or a history of one or more recurring or chronic porphyric symptoms, and/or a subject who carries a genetic alteration (e.g., a mutation) in a gene encoding an enzyme of the heme biosynthetic pathway, and a subject who carries a genetic alteration, e.g., a mutation. known to be associated with porphyria.

In embodiments, the alteration, e.g., the mutation, makes an individual susceptible to an acute attack (e.g., upon exposure to a precipitating factor, e.g., a drug, dieting or other precipitating factor, e.g., a precipitating factor as disclosed herein). In embodiments, the alteration, e.g., the mutation, is associated with elevated levels of a porphyrin or a porphyrin precursor (e.g., ALA and/or PBG). In embodiments, the alteration, e.g., the mutation, is associated with chronic pain (e.g., chronic neuropathic pain) and/or neuropathy (e.g., progressive neuropathy). In embodiments, the, the alteration, e.g., the mutation, is associated with changes in EMG and/or nerve conduction velocities.

In embodiments, the alteration is a mutation in the ALAS1 gene. In embodiments, the alteration is a mutation in the ALAS1 gene promoter, or in regions upstream or downstream from the ALAS1 gene. In embodiments, the alteration is a mutation in transcription factors or other genes that interact with ALAS1. In embodiments, the alteration is an alteration, e.g., a mutation, in a gene that encodes an enzyme in the heme biosynthetic pathway.

In some embodiments, the subject has an genetic alteration as described herein (e.g., a genetic mutation known to be associated with a porphyria). In some such embodiments, the subject has an elevated level (e.g., urine or plasma level) of ALA and/or PBG. In some such embodiments, the subject does not have an elevated level of ALA and/or PBG. In embodiments, the subject has a genetic alteration as described herein and has other symptoms, e.g., chronic pain, EMG changes, changes in nerve conduction velocity, and/or other symptoms associated with a porphyria. In embodiments, the subject has a genetic alteration but does not suffer from acute attacks.

In embodiments, the subject has a mutation associated with AIP, HCP, VP, or ADP.

In some embodiments, the porphyria is AIP. In some such embodiments, the subject has an alteration, e.g., at least one mutation, in the PBG deaminase gene. Many PBG deaminase mutations are known in the art, for example, as reported in Hrdinka, M. et al. Physiological Research, 55 (Suppl2):S119-136 (2006). In some embodiments, the subject is heterozygous for a PBG deaminase mutation. In other embodiments, the subject is homozygous for a PBG deaminase mutation. A homozygous subject may carry two identical mutations or two different mutations in the PBG deaminase gene.

In some embodiments, the porphyria is HCP. In some such embodiments, the subject has an alteration, e.g., at least one mutation, in the gene that encodes the enzyme coproporphyrinogen III oxidase.

In some embodiments, the porphyria is VP. In some such embodiments, the subject has an alteration, e.g., at least one mutation, in the gene that encodes protoporphrinogen oxidase.

In embodiments, the porphyria is ADP, e.g., autosomal recessive ADP. In some such embodiments, the subject has an alteration, e.g., at least one mutation, in the gene that encodes ALA deydratase.

Methods of treatment provided herein may serve to ameliorate one or more symptoms associated with porphyria, to reduce the frequency of attacks associated with porphyria, to reduce the likelihood that an attack of one or more symptoms associated with porphyria will occur upon exposure to a precipitating factor, or to reduce the risk of developing conditions associated with porphyria (e.g., neuropathy (e.g., progressive neuropathy), hepatocellular cancer). Additionally, the methods provided herein may serve to decrease the level of one or more porphyrin precursors, porphyrins and/or related porphyrin products or metabolites. The level of a porphyrin precursor or a porphyrin may be measured in any biological sample, such as, e.g., urine, blood, feces, cerebrospinal fluid, or a tissue sample. The sample may be present within a subject or may be obtained or extracted from the subject. In some embodiments, the porphyria is AIP, and the level of PBG and/or ALA is decreased. In some embodiments, the porphyrin product or metabolite is porphobilin, porphobilinogen, or uroporphyrin. A decrease in the level of a porphyrin product or metabolite may be measured using any method known in the art. For example, the level of PBG and/or ALA in urine or plasma may be assessed, using the Watson-Schwartz test, ion exchange chromatography, or high-performance liquid chromatography—mass spectrometry. See, e.g., Thunell (1993).

Methods described herein may also serve to reduce chronically elevated levels of porphyrin precursors (e.g., ALA and/or PBG) in subjects suffering from a porphyria (e.g., an acute hepatic porphyria, e.g., AIP) or at risk for developing a porphyria. Methods for assessing plasma and urine levels (e.g., chronically elevated levels) of porphyrin precursors include, e.g., HPLC-mass spectrometry and ion-exchange chromatography. The levels of porphyrin precursors may be expressed as the level relative to another protein or compound, e.g., creatinine. See, e.g., Floderus, Y. et al, Clinical Chemistry, 52(4): 701-707, 2006; Sardh et al., Clinical Pharmacokinetics, 46(4): 335-349, 2007

A “precipitating factor” as used herein, refers to an endogenous or exogenous factor that may induce an acute attack of one or more symptoms associated with porphyria. Precipitating factors include fasting (or other forms of reduced or inadequate caloric intake, due to crash diets, long-distance athletics, etc.), metabolic stresses (e.g., infections, surgery, international air travel, and psychological stress), endogenous hormones (e.g., progesterone), cigarette smoking, lipid-soluble foreign chemicals (including, e.g., chemicals present in tobacco smoke, certain prescription drugs, organic solvents, biocides, components in alcoholic beverages), endocrine factors (e.g., reproductive hormones (women may experience exacerbations during the premenstrual period), synthetic estrogens, progesterones, ovulation stimulants, and hormone replacement therapy). See, for example, Thunell (1993). Common precipitating factors include cytochrome P450 inducing drugs and phenobarbitol.

Symptoms associated with porphyria may include abdominal pain or cramping, headaches, effects caused by nervous system abnormalities, and light sensitivity, causing rashes, blistering, and scarring of the skin (photodermatitis). In certain embodiments, the porphyria is AIP. Symptoms of AIP include gastrointestinal symptoms (e.g., severe and poorly localized abdominal pain, nausea/vomiting, constipation, diarrhea, ileus), urinary symptoms (dysuria, urinary retention/incontinence, or dark urine), neurologic symptoms (e.g., sensory neuropathy, motor neuropathy (e.g., affecting the cranial nerves and/or leading to weakness in the arms or legs), seizures, neuropathic pain, progressive neuropathy, headaches, neuropsychiatric symptoms (e.g., mental confusion, anxiety, agitation, hallucination, hysteria, delirium, apathy, depression, phobias, psychosis, insomnia, somnolence, coma), autonomic nervous system involvement (resulting e.g., in cardiovascular sysmptoms such as tachycardia, hypertension, and/or arrhythmias, as well as other symptoms, such as, e.g., increased circulating catecholamine levels, sweating, restlessness, and/or tremor), dehydration, and electrolyte abnormalities.

In some embodiments, an iRNA targeting ALAS1 is administered together with (e.g., before, after, or concurrent with) another treatment that may serve to alleviate one or more of the above symptoms. For example, abdominal pain may be treated, e.g., with narcotic analgesics, seizures may be treated, e.g., with anti-seizure medications, nausea/vomiting may be treated, e.g., with phenothiazines, and tachycardia/hypertension may be treated, e.g., with beta blockers.

The term “decrease” (or “increase”) is intended to refer to a measurable change, e.g., a statistically significant change. The change may be, for example, at least 5%, 10%, 20%, 30%, 40%, 50% or more change (e.g., decrease (or increase) relative to a reference value, e.g., a reference where no iRNA is provided).

The invention further relates to the use of an iRNA or a pharmaceutical composition thereof, e.g., for treating a disorder related to ALAS1 expression, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating the disorder. In one embodiment, the iRNA or pharmaceutical composition thereof can be administered in conjunction with a heme product (e.g., hemin, heme arginate, or heme albumin, as described herein) and/or in conjunction with intravenous glucose infusions. In some embodiments, the iRNA or pharmaceutical composition thereof is used prophylactically, e.g., to prevent or ameliorate symptoms of an anticipated attack of acute porphyria. The prophylactic use may be timed according to the exposure or anticipated exposure of the subject to a precipitating factor. As described herein, a precipitating factor may be any endogenous or exogenous factor known to precipitate an acute attack. For example, the premenstrual phase is an endogenous precipitating factor, and a cytochrome P450 inducing drug is an exogenous precipitating factor.

The effective amount for the treatment of a disorder related to ALAS1 expression (e.g., a porphyria such as AIP) depends on the type of disorder to be treated, the severity of the symptoms, the subject being treated, the sex, age and general condition of the subject, the mode of administration and so forth. For any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using routine experimentation. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an iRNA targeting ALAS1 or pharmaceutical composition thereof, “effective against” a disorder related to ALAS1 expression indicates that administration in a clinically appropriate manner results in a beneficial effect, e.g., for an individual patient or for at least a fraction of patients, e.g., a statistically significant fraction of patients. Beneficial effects include, e.g., prevention of or reduction of symptoms or other effects. For example, beneficial effects include, e.g., an improvement (e.g., decrease in the severity or frequency) of symptoms, a reduction in the severity or frequency of attacks, a reduced risk of developing associated disease (e.g., neuropathy (e.g., progressive neuropathy), hepatocellular cancer), an improved ability to tolerate a precipitating factor, an improvement in quality of life, a reduction in the expression of ALAS1, a reduction in a level (e.g., a plasma or urine level) of a porphyrin or a porphyrin precursor (e.g., ALA and/or PBG) or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disorder.

A treatment or preventive effect is evident when there is an improvement, e.g., a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, e.g., at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker (e.g., plasma or urinary ALA or PBG) or symptom is observed.

Patients can be administered a therapeutic amount of iRNA. The therapeutic amount can be, e.g., 0.05-50 mg/kg. For example, the therapeutic amount can be 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, or 2.5, 3.0, 3.5, 4.0, 4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg/kg dsRNA.

In some embodiments, the iRNA is formulated as a lipid formulation, e.g., an LNP formulation as described herein. In some such embodiments, the therapeutic amount is 0.05-5 mg/kg, e.g., 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg/kg dsRNA. In some embodiments, the lipid formulation, e.g., LNP formulation, is administered intravenously.

In some embodiments, the iRNA is administered by intravenous infusion over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.

In some embodiments, the iRNA is in the form of a GalNAc conjugate as described herein. In some such embodiments, the therapeutic amount is 0.5-50 mg, e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg/kg dsRNA. In some embodiments, the GalNAc conjugate is administered subcutaneously.

In some embodiments, the administration is repeated, for example, on a regular basis, such as, daily, biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer.

In some embodiments, the iRNA agent is administered in two or more doses. In some embodiments, the number or amount of subsequent doses is dependent on the achievement of a desired effect, e.g., suppression of a ALAS gene, reduction of a level of a porphyrin or porphyrin precursor (e.g., ALA and/or PBG), or the achievement of a therapeutic or prophylactic effect, e.g., reduction or prevention of one or more symptoms associated with porphyria (e.g., pain, e.g., neuropathic pain), and/or prevention of attacks or reduction in the frequency and/or severity of attacks associated with porphyria.

In some embodiments, the iRNA agent is administered according to a schedule. For example, the iRNA agent may be administered once per week, twice per week, three times per week, four times per week, or five times per week. In some embodiments, the schedule involves regularly spaced administrations, e.g., hourly, every four hours, every six hours, every eight hours, every twelve hours, daily, every 2 days, every 3 days, every 4 days, every 5 days, weekly, biweekly, or monthly. In embodiments, the iRNA agent is administered weekly or biweekly to achieve a desired effect, e.g., to decrease the level of ALA and/or PBG, to decrease pain, and/or to prevent acute attacks.

In embodiments, the schedule involves closely spaced administrations followed by a longer period of time during which the agent is not administered. For example, the schedule may involve an initial set of doses that are administered in a relatively short period of time (e.g., about every 6 hours, about every 12 hours, about every 24 hours, about every 48 hours, or about every 72 hours) followed by a longer time period (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks) during which the iRNA agent is not administered. In one embodiment, the iRNA agent is initially administered hourly and is later administered at a longer interval (e.g., daily, weekly, biweekly, or monthly). In another embodiment, the iRNA agent is initially administered daily and is later administered at a longer interval (e.g., weekly, biweekly, or monthly). In certain embodiments, the longer interval increases over time or is determined based on the achievement of a desired effect. In a specific embodiment, the iRNA agent is administered once daily during an acute attack, followed by weekly dosing starting on the eighth day of administration. In another specific embodiment, the iRNA agent is administered every other day during a first week followed by weekly dosing starting on the eighth day of administration.

In one embodiment, the iRNA agent is administered to prevent or reduce the severity or frequency of recurring attacks, e.g., cyclical attacks associated with a precipitating factor. In some embodiments, the precipitating factor is the menstrual cycle. In some embodiments, the iRNA is administered repeatedly, e.g., at regular intervals to prevent or reduce the severity or frequency of recurring attacks, e.g., cyclical attacks associated with a precipitating factor, e.g., the menstrual cycle, e.g., a particular phase of the menstrual cycle, e.g., the luteal phase. In some embodiments, the iRNA is administered during a particular phase of the menstrual cycle or based on hormone levels of the patient being treated (e.g., based on hormone levels that are associated with a particular phase of the menstrual cycle). In some embodiments, the iRNA is administered on one or more particular days of the menstrual cycle, e.g., on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or on day 28 (or later day for subjects who have a longer menstrual cycle). In some embodiments, the iRNA is administered during the luteal phase, e.g., on one or more days between days 14-28 of the menstrual cycle (or later, in subjects who have a menstrual cycle longer than 28 days). In some embodiments, ovulation of the subject is assessed (e.g., using a blood or urine test that detects a hormone associated with ovulation, e.g., LH) and the iRNA is administered at a predetermined interval after ovulation. In some embodiments, the iRNA is administered immediately after ovulation. In some embodiments, the iRNA is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days after ovulation. Any of these schedules may optionally be repeated for one or more iterations. The number of iterations may depend on the achievement of a desired effect, e.g., the suppression of a ALAS1 gene and/or the achievement of a therapeutic or prophylactic effect, e.g., reduce or prevent one or more symptoms associated with porphyria, to reduce the frequency of attacks associated with porphyria.

In some embodiments, an initial dose of the iRNA agent is administered and the level of ALA or PBG is tested, e.g., 1-48 hours, e.g., 2, 4, 8, 12, or 24 hours following administration of the initial dose. In some embodiments, if the level of ALA and/or PBG has decreased (e.g., to achieve a predetermined reduction, e.g., a normalization), and/or if the symptoms associated with porphyria (e.g., pain) have improved (e.g., such that the patient is asymptomatic), no further dose is administered, whereas if the level of ALA and/or PBG has not decreased (e.g., has not achieved a predetermined reduction, e.g., has not normalized), a further dose of ALA or PBG is administered. In some embodiments, the further dose is administered 12, 24, 36, 48, 60, or 72 hours after the initial dose. In some embodiments, if the initial dose is not effective to decrease the level of ALA and/or PBG, the further dose is modified, e.g., increased to achieve a desired decrease (e.g., a predetermined reduction, e.g., a normalization) in ALA or PBG levels.

In some embodiments, the predetermined reduction is a decrease of at least 10%, 20%, 30%, 40%, or 50%. In some embodiments, the predetermined reduction is a reduction that is effective to prevent or ameliorate symptoms, e.g., pain, prodromal symptoms, or recurring attacks.

In some embodiments, the predetermined reduction is a reduction of at least 1, 2, 3, or more standard deviations, wherein the standard deviation is determined based on the values from a reference sample, e.g., a reference sample as described herein.

In some embodiments, the predetermined reduction is a reduction that brings the level of the porphyrin or porphyrin precursor to a level that is less than, or to a level that is less than or equal to, a reference value (e.g., a reference value as described herein).

As used herein, a “normalization” in ALA or PBG levels (or a “normal” or “normalized” level) refers to a level (e.g., a urine and/or plasma level) of either ALA, or PBG, or both, that is within the expected range for a healthy individual, an individual who is asymptomatic (e.g., an individual who does not experience pain and/or suffer from neuropathy), or an individual who does not have a mutation associated with a porphyria. For example, in some embodiments, a normalized level is within two standard deviations of the normal mean. In some embodiments, a normalized level is within normal reference limits, e.g., within the 95% confidence interval for an appropriate control sample, e.g., a sample of healthy individuals or individuals who do not carry a gene mutation associated with a porphyria. In some embodiments, the ALA and/or PBG level of the subject (e.g., the urine and/or plasma ALA and/or PBG level) is monitored at intervals, a further dose of the iRNA agent is administered when the level increases above the reference value

Administration of the iRNA may reduce ALAS1 mRNA or protein levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% or more. Administration of the iRNA may reduce levels of products associated with ALAS1 gene expression, e.g., levels of one or more porphyrins or porphyrin precursors (e.g., the level of ALA and/or PBG). Administration of the iRNA agent may also inhibit or prevent the upregulation of ALAS1 mRNA or protein levels during an acute attack of AIP.

Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion dose, and monitored for adverse effects, such as an allergic reaction, or for elevated lipid levels or blood pressure. In another example, the patient can be monitored for unwanted effects.

Methods for Modulating Expression of an ALAS1 Gene

In yet another aspect, the invention provides a method for modulating (e.g., inhibiting or activating) the expression of an ALAS1 gene, e.g., in a cell or in a subject. In some embodiments, the cell is ex vivo, in vitro, or in vivo. In some embodiments, the cell is an erythroid cell or a hepatocyte. In some embodiments, the cell is in a subject (e.g., a mammal, such as, for example, a human). In some embodiments, the subject (e.g., the human) is at risk, or is diagnosed with a disease related to ALAS1 expression, as described above.

In one embodiment, the method includes contacting the cell with an iRNA as described herein, in an amount effective to decrease the expression of an ALAS1 gene in the cell. “Contacting,” as used herein, includes directly contacting a cell, as well as indirectly contacting a cell. For example, a cell within a subject (e.g., an erythroid cell or a liver cell, such as a hepatocyte) may be contacted when a composition comprising an iRNA is administered (e.g., intravenously or subcutaneously) to the subject.

The expression of an ALAS1 gene may be assessed based on the level of expression of an ALAS1 mRNA, an ALAS1 protein, or the level of a parameter functionally linked to the level of expression of an ALAS1 gene (e.g., the level of a porphyrin or the incidence or severity of a symptom related to a porphyria). In some embodiments, the expression of ALAS1 is inhibited by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, the iRNA has an IC₅₀ in the range of 0.001-0.01 nM, 0.001-0.10 nM, 0.001-1.0 nM, 0.001-10 nM, 0.01-0.05 nM, 0.01-0.50 nM, 0.02-0.60 nM, 0.01-1.0 nM, 0.01-1.5 nM, 0.01-10 nM. The IC₅₀ value may be normalized relative to an appropriate control value, e.g., the IC₅₀ of a non-targeting iRNA.

In some embodiments, the method includes introducing into the cell an iRNA as described herein and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of an ALAS1 gene, thereby inhibiting the expression of the ALAS1 gene in the cell.

In one embodiment, the method includes administering a composition described herein, e.g., a composition comprising an iRNA that targets ALAS1, to the mammal such that expression of the target ALAS1 gene is decreased, such as for an extended duration, e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, or four weeks or longer. In some embodiments, the decrease in expression of ALAS1 is detectable within 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours of the first administration.

In another embodiment, the method includes administering a composition as described herein to a mammal such that expression of the target ALAS1 gene is increased by e.g., at least 10% compared to an untreated animal. In some embodiments, the activation of ALAS1 occurs over an extended duration, e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, four weeks, or more. Without wishing to be bound by theory, an iRNA can activate ALAS1 expression by stabilizing the ALAS1 mRNA transcript, interacting with a promoter in the genome, and/or inhibiting an inhibitor of ALAS1 expression.

The iRNAs useful for the methods and compositions featured in the invention specifically target RNAs (primary or processed) of an ALAS1 gene. Compositions and methods for inhibiting the expression of an ALAS1 gene using iRNAs can be prepared and performed as described elsewhere herein.

In one embodiment, the method includes administering a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the ALAS1 gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration.

In certain embodiments, the compositions are administered by intravenous infusion or injection. In some such embodiments, the compositions comprise a lipid formulated siRNA (e.g., an LNP formulation, such as an LNP11 formulation) for intravenous infusion. In particular embodiments, such compositions may be used to treat acute attacks of porphyria and/or for prophylaxis (e.g., to decrease the severity or frequency of attacks).

In other embodiments, the compositions are administered subcutaneously. In some such embodiments, the compositions comprise an iRNA conjugated to a GalNAc ligand. In particular embodiments, such compositions may be used to treat acute attacks of porphyria or for prophylaxis (e.g., to decrease the severity or frequency of attacks).

Methods for Decreasing a Level of a Porphyrin or Porphyrin Precursor

In another aspect, the invention provides a method for decreasing a level of a porphyrin or a porphyrin precursor, e.g., in a cell or in a subject.

In some embodiments, the cell is ex vivo, in vitro, or in vivo. In some embodiments, the cell is an erythroid cell or a hepatocyte. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is in a subject (e.g., a mammal, such as, for example, a human).

In some embodiments, the subject (e.g., the human) is at risk, or is diagnosed with a porphyria, as described herein. In some embodiments, the method is effective to treat a porphyria as described herein (e.g., by ameliorating one or more symptoms associated with a porphyria, reducing the frequency of attacks associated with a porphyria, reducing the likelihood that an attack of one or more symptoms associated with porphyria will occur upon exposure to a precipitating factor, or reducing the risk of developing conditions associated with a porphyria (e.g., neuropathy (e.g., progressive neuropathy), hepatocellular cancer). In one embodiment, the method includes contacting the cell with an RNAi, as described herein, in an amount sufficient to decrease the level of the porphyrin or porphyrin precursor (e.g., ALA or PBG) in the cell, or in another related cell or group of cells, or in the subject. “Contacting,” as used herein, includes directly contacting a cell, as well as indirectly contacting a cell. For example, a cell within a subject (e.g., an erythroid cell or a liver cell, such as a hepatocyte) may be contacted when a composition comprising an RNAi is administered (e.g., intravenously or subcutaneously) to the subject. “Another related cell or group of cells,” as used herein, includes any cell or group of cells in which the level of the porphyrin or porphyrin precursor decreases as a result of the contacting. For example, the cell may be part of a tissue present within a subject (e.g., a liver cell present within a subject), and contacting the cell within the subject (e.g., contacting one or more liver cells present within a subject) with the RNAi may result in a decrease in the level of the porphyrin or porphyrin precursor in another related cell or group of cells (e.g., nerve cells of the subject), or in a tissue or fluid of the subject (e.g., in the urine, blood, plasma, or cerebrospinal fluid of the subject).

In some embodiments, the porphyrin or porphyrin precursor is selected from the group consisting of δ-aminolevulinic acid (ALA), porphopilinogen (PBG), hydroxymethylbilane (INMB), uroporphyrinogen III, coproporphyrinogen III, protoporphrinogen IX, and protoporphyrin IX In some embodiments the porphyrin precursor is ALA. In some embodiments, the porphyrin precursor is PBG. In some embodiments, the method decreases the level of ALA and PBG. The level of a porphyrin or a porphyrin precursor may be measured as described herein and as known in the art.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES Example 1. siRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Oligonucleotide Synthesis.

All oligonucleotides are synthesized on an AKTAoligopilot synthesizer. Commercially available controlled pore glass solid support (dT-CPG, 500{acute over (Å)}, Prime Synthesis) and RNA phosphoramidites with standard protecting groups, 5′-O-dimethoxytrityl N6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, and 5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies) were used for the oligonucleotide synthesis. The 2′-F phosphoramidites, 5′-O-dimethoxytrityl-N4-acetyl-2′-fluro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite and 5′-O-dimethoxytrityl-2′-fluro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite are purchased from (Promega). All phosphoramidites are used at a concentration of 0.2M in acetonitrile (CH₃CN) except for guanosine which is used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recycling time of 16 minutes is used. The activator is 5-ethyl thiotetrazole (0.75M, American International Chemicals); for the PO-oxidation iodine/water/pyridine is used and for the PS-oxidation PADS (2%) in 2,6-lutidine/ACN (1:1 v/v) is used.

3′-ligand conjugated strands are synthesized using solid support containing the corresponding ligand. For example, the introduction of cholesterol unit in the sequence is performed from a hydroxyprolinol-cholesterol phosphoramidite. Cholesterol is tethered to trans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain a hydroxyprolinol-cholesterol moiety. 5′-end Cy-3 and Cy-5.5 (fluorophore) labeled iRNAs are synthesized from the corresponding Quasar-570 (Cy-3) phosphoramidite are purchased from Biosearch Technologies. Conjugation of ligands to 5′-end and or internal position is achieved by using appropriately protected ligand-phosphoramidite building block. An extended 15 min coupling of 0.1 M solution of phosphoramidite in anhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activator to a solid-support-bound oligonucleotide. Oxidation of the internucleotide phosphite to the phosphate is carried out using standard iodine-water as reported (1) or by treatment with tert-butyl hydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation wait time conjugated oligonucleotide. Phosphorothioate is introduced by the oxidation of phosphite to phosphorothioate by using a sulfur transfer reagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucage reagent. The cholesterol phosphoramidite is synthesized in house and used at a concentration of 0.1 M in dichloromethane. Coupling time for the cholesterol phosphoramidite is 16 minutes.

Deprotection I (Nucleobase Deprotection)

After completion of synthesis, the support is transferred to a 100 mL glass bottle (VWR). The oligonucleotide is cleaved from the support with simultaneous deprotection of base and 30 phosphate groups with 80 mL of a mixture of ethanolic ammonia [ammonia: ethanol (3:1)] for 6.5 h at 55° C. The bottle is cooled briefly on ice and then the ethanolic ammonia mixture is filtered into a new 250-mL bottle. The CPG is washed with 2×40 mL portions of ethanol/water (1:1 v/v). The volume of the mixture is then reduced to ˜30 mL by roto-vap. The mixture is then frozen on dry ice and dried under vacuum on a speed vac.

Deprotection II (Removal of 2′-TBDMS Group)

The dried residue is resuspended in 26 mL of triethylamine, triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6) and heated at 60° C. for 90 minutes to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reaction is then quenched with 50 mL of 20 mM sodium acetate and the pH is adjusted to 6.5. Oligonucleotide is stored in a freezer until purification.

Analysis

The oligonucleotides are analyzed by high-performance liquid chromatography (HPLC) prior to purification and selection of buffer and column depends on nature of the sequence and or conjugated ligand.

HPLC Purification

The ligand-conjugated oligonucleotides are purified by reverse-phase preparative HPLC. The unconjugated oligonucleotides are purified by anion-exchange HPLC on a TSK gel column packed in house. The buffers are 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractions containing full-length oligonucleotides are pooled, desalted, and lyophilized. Approximately 0.15 OD of desalted oligonucleotides are diluted in water to 150 μL and then pipetted into special vials for CGE and LC/MS analysis. Compounds are then analyzed by LC-ESMS and CGE.

siRNA Preparation

For the general preparation of siRNA, equimolar amounts of sense and antisense strand are heated in 1×PBS at 95° C. for 5 min and slowly cooled to room temperature. Integrity of the duplex is confirmed by HPLC analysis.

Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 1.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) A Adenosine-3′-phosphate Ab beta-L-adenosine-3′-phosphate Abs beta-L-adenosine-3′-phosphorothioate Af 2′-fluoroadenosine-3′-phosphate Afs 2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioate C cytidine-3′-phosphate Cb beta-L-cytidine-3′-phosphate Cbs beta-L-cytidine-3′-phosphorothioate Cf 2′-fluorocytidine-3′-phosphate Cfs 2′-fluorocytidine-3′-phosphorothioate (Chd) 2′-O-hexadecyl-cytidine-3′-phosphate (Chds) 2′-O-hexadecyl-cytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate G guanosine-3′-phosphate Gb beta-L-guanosine-3′-phosphate Gbs beta-L-guanosine-3′-phosphorothioate Gf 2′-fluoroguanosine-3′-phosphate Gfs 2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioate T 5′-methyluridine-3′-phosphate Tb beta-L-thymidine-3′-phosphate Tbs beta-L-thymidine-3′-phosphorothioate Tf 2′-fluoro-5-methyluridine-3′-phosphate Tfs 2′-fluoro-5-methyluridine-3′-phosphorothioate Ts 5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Ub beta-L-uridine-3′-phosphate Ubs beta-L-uridine-3′-phosphorothioate Uf 2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioate (Uhd) 2′-O-hexadecyl-uridine-3′-phosphate (Uhds) 2′-O-hexadecyl-uridine-3′-phosphorothioate Us uridine-3′-phosphorothioate N any nucleotide (G, A, C, T or U) a 2′-O-methyladenosine-3′-phosphate as 2′-O-methyladenosine-3′-phosphorothioate c 2′-O-methylcytidine-3′-phosphate cs 2′-O-methylcytidine-3′-phosphorothioate g 2′-O-methylguanosine-3′-phosphate gs 2′-O-methylguanosine-3′-phosphorothioate t 2′-O-methyl-5-methyluri dine-3′-phosphate ts 2′-O-methyl-5-methyluridine-3′-phosphorothioate u 2′-O-methyluridine-3′-phosphate us 2′-O-methyluridine-3′-phosphorothioate dA 2′-deoxyadenosine-3′-phosphate dAs 2′-deoxyadenosine-3′-phosphorothioate dC 2′-deoxycytidine-3′-phosphate dCs 2′-deoxycytidine-3′-phosphorothioate dG 2′-deoxyguanosine-3′-phosphate dGs 2′-deoxyguanosine-3′-phosphorothioate dT 2′-deoxythymidine dTs 2′-deoxythymidine-3′-phosphorothioate dU 2′-deoxyuridine s phosphorothioate linkage L96¹ N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4- hydroxyprolinol Hyp-(GalNAc-alkyl)3 (Aeo) 2′-O-methoxyethyladenosine-3′-phosphate (Aeos) 2′-O-methoxyethyladenosine-3′-phosphorothioate (Geo) 2′-O-methoxyethylguanosine-3′-phosphate (Geos) 2′-O-methoxyethylguanosine-3′-phosphorothioate (Teo) 2′-O-methoxyethyl-5-methyluridine-3′-phosphate (Teos) 2′-O-methoxyethyl-5-methyluridine-3′-phosphorothioate (m5Ceo) 2′-O-methoxyethyl-5-methylcytidine-3′-phosphate (m5Ceos) 2′-O-methoxyethyl-5-methylcytidine-3′-phosphorothioate ¹The chemical structure of L96 is as follows:

Example 2. ALAS1 siRNA Design and Synthesis Experimental Methods Bioinformatics Transcripts

siRNA design was carried out to identify siRNAs targeting human, rhesus (Macaca mulatta), mouse, and rat ALAS1 transcripts annotated in the NCBI Gene database (http://www.ncbi.nlm.nih.gov/gene/). Design used the following transcripts from the NCBI RefSeq collection: Human—NM_000688.4 (see FIG. 3), NM_199166.1; Rhesus—XM_001090440.2, XM_001090675.2; Mouse—NM_020559.2; Rat—NM_024484.2. Due to high primate/rodent sequence divergence, siRNA duplexes were designed in several separate batches, including but not limited to batches containing duplexes matching human and rhesus transcripts only; human, rhesus, mouse, and rat transcripts only; and mouse and rat transcripts only. Most siRNA duplexes were designed that shared 100% identity the listed human transcript and other species transcripts considered in each design batch (above). In some instances, (see Table 8) mismatches between duplex and mRNA target were allowed at the first antisense (last sense) position when the antisense strand:target mRNA complementary basepair was a GC or CG pair. In these cases, duplexes were designed with UA or AU pairs at the first antisense:last sense pair. Thus the duplexes maintained complementarity but were mismatched with respect to target (U:C, U:G, A:C, or A:G). Eighteen of these “UA-swap” duplexes were designed as part of the human/rhesus/mouse/rat set (see duplexes in Table 8 with “C19U”, “G19U”, “C19A”, or “G19A” labels in the Position column).

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were then selected that lacked repeats longer than 7 nucleotides. These 1510 candidate human/rhesus, 114 human/rhesus/mouse/rat, and 717 mouse/rat siRNAs were used in comprehensive searches against the appropriate transcriptomes (defined as the set of NM_ and XM_records within the human, rhesus, dog, mouse, or rat NCBI Refseq sets) using an exhaustive “brute-force” algorithm implemented in the python script ‘BruteForce.py’. The script next parsed the transcript-oligo alignments to generate a score based on the position and number of mismatches between the siRNA and any potential ‘off-target’ transcript. The off-target score is weighted to emphasize differences in the ‘seed’ region of siRNAs, in positions 2-9 from the 5′ end of the molecule. Each oligo-transcript pair from the brute-force search was given a mismatch score by summing the individual mismatch scores; mismatches in the position 2-9 were counted as 2.8, mismatches in the cleavage site positions 10-11 were counted as 1.2, and mismatches in region 12-19 counted as 1.0. An additional off-target prediction was carried out by comparing the frequency of heptamers and octomers derived from 3 distinct, seed-derived hexamers of each oligo. The hexamers from positions 2-7 relative to the 5′ start is used to create 2 heptamers and one octomer. We create ‘heptamer1’ by adding a 3′ A to the hexamer; we create heptamer2 by adding a 5′ A to the hexamer; we create the octomer by adding an A to both 5′ and 3′ ends of the hexamer. The frequency of octomers and heptamers in the human, rhesus, mouse, or rat 3′UTRome (defined as the subsequence of the transcriptome from NCBI's Refseq database where the end of the coding region, the ‘CDS’, is clearly defined) was pre-calculated. The octomer frequency was normalized to the heptamer frequency using the median value from the range of octomer frequencies. A ‘mirSeedScore’ was then calculated by calculating the sum of ((3×normalized octomer count)+(2×heptamer2 count)+(1×heptamer1 count)).

Both siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand. We then selected duplexes whose antisense oligos lacked GC at the first position, lacked G at both positions 13 and 14, and had 3 or more Us or As in the seed region (characteristics of duplexes with high predicted efficacy)

Candidate GalNac-conjugated duplexes, 21 and 23 nucleotides long on the sense and antisense strands respectively, were designed by extending antisense 19mers 4 additional nucleotides in the 3′ direction (preserving perfect complementarity with the target transcript). The sense strand was specified as the reverse complement of the first 21 nucleotides of the antisense 23mer. Duplexes were selected that maintained perfect matches to all selected species transcripts across all 23 nucleotides.

siRNA Sequence Selection

A total of 90 sense and 90 antisense derived human/rhesus, 40 sense and 40 antisense derived human/rhesus/mouse/mouse/rat, and 40 sense and 40 antisense derived mouse/rat siRNA 19mer oligos were synthesized and formed into duplexes. A total of 45 sense and 45 antisense derived human/rhesus 21/23mer oligos were synthesized to yield 45 GalNac-conjugated duplexes.

The sequences of the sense and antisense strands of the modified duplexes are shown in Table 2, and the sequences of the sense and antisense strands of the unmodified duplexes are shown in Table 3.

Synthesis of ALAS1 Sequences

ALAS1 sequences were synthesized on MerMade 192 synthesizer at either 1 or 0.2 umol scale. Single strands were made with 2′O-methyl modifications for in vitro screening using transfection reagents. 3′ GalNAc conjugates were made with sequences containing 2′F and 2′-O-methyl modifications on the sense strand in the 21-23 mer designs for free uptake in cells. For all the 21mer sequences in the list, ‘endolight’ chemistry was applied as detailed below.

-   -   All pyrimidines (cytosine and uridine) in the sense strand         contained 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U)     -   In the antisense strand, pyrimidines adjacent to (towards 5′         position) ribo A nucleoside were replaced with their         corresponding 2-O-Methyl nucleosides     -   A two base dTsdT extension at 3′ end of both sense and anti         sense sequences was introduced     -   The sequence file was converted to a text file to make it         compatible for loading in the MerMade 192 synthesis software

For GalNAc conjugated sense strands and complementary antisense sequences, 2′F and other modified nucleosides were introduced in combination with ribo with 2′O-Methyl nucleosides. The synthesis was performed on a GalNAc modified CPG support for the sense strand and CPG modified with universal support on the antisense sequence.

Synthesis, Cleavage and Deprotection:

The synthesis of ALAS1 sequences used solid supported oligonucleotide synthesis using phosphoramidite chemistry. For 21 mer endolight sequences, a deoxy thymidine CPG was used as the solid support while for the GalNAc conjugates, GalNAc solid support for sense strand and an universal CPG for the antisense strand were used.

The synthesis of the above sequences was performed at either 1 or 0.2 um scale in 96 well plates. The amidite solutions were prepared at 0.1M concentration and ethyl thio tetrazole (0.6M in Acetonitrile) was used as activator.

The synthesized sequences were cleaved and deprotected in 96 well plates, using methylamine in the first step and fluoride reagent in the second step. For GalNAc and 2′F nucleoside containing sequences, deprotection conditions were modified. Sequences after cleavage and deprotection were precipitated using acetone:ethanol (80:20) mix and the pellet were re-suspended in 0.2M sodium acetate buffer. Samples from each sequence were analyzed by LC-MS to confirm the identity, UV for quantification and a selected set of samples by IEX chromatography to determine purity.

PURIFICATION and desalting:

ALAS1 sequences were precipitated and purified on AKTA Purifier system using Sephadex column. The ALAS1ess was run at ambient temperature. Sample injection and collection was performed in 96 well (1.8 mL-deep well) plates. A single peak corresponding to the full length sequence was collected in the eluent. The desalted ALAS1 sequences were analyzed for concentration (by UV measurement at A260) and purity (by ion exchange HPLC). The complementary single strands were then combined in a 1:1 stoichiometric ratio to form siRNA duplexes.

TABLE 2 Human ALAS1 Modified Single Strands and Duplex Sequences SEQ ID SEQ ID NO: Position on NO: (anti- transcript Duplex Sense Sequence Antisense Sequence (sense) sense) NM_000688.4 Name (5′-3′) (5′-3′) 2 3 522-540 AD-55078.2 cuccGGccAGuGAGAAAGAdTsdT UCUUUCUcACUGGCCGGAGdTsdT 4 5 669-687 AD-55084.2 uGGcAGcAcAGAuGAAucAdTsdT UGAUUcAUCUGUGCUGCcAdTsdT 6 7 790-808 AD-55090.2 cAGuGuGGuuAGuGuGAAAdTsdT UUUcAcACuAACcAcACUGdTsdT 8 9 853-871 AD-55096.2 cAucAuGcAAAAGcAAAGAdTsdT UCUUUGCUUUUGcAUGAUGdTsdT 10 11 876-894 AD-55102.2 AAAGAGuGucucAucuucudTsdT AGAAGAUGAGAcACUCUUUdTsdT 12 13 877-895 AD-55106.2 AAGAGuGucucAucuucuudTsdT AAGAAGAUGAGAcACUCUUdTsdT 14 15 914-932 AD-55111.2 ucuGuuuccAcuuuucAGudTsdT ACUGAAAAGUGGAAAcAGAdTsdT 16 17 923-941 AD-55073.2 AcuuuucAGuAuGAucGuudTsdT AACGAUcAuACUGAAAAGUdTsdT 18 19 926-944 AD-55079.2 uuucAGuAuGAucGuuucudTsdT AGAAACGAUcAuACUGAAAdTsdT 20 21 927-945 AD-55085.2 uucAGuAuGAucGuuucuudTsdT AAGAAACGAUcAuACUGAAdTsdT 22 23 928-946 AD-55091.2 ucAGuAuGAucGuuucuuudTsdT AAAGAAACGAUcAuACUGAdTsdT 24 25 932-950 AD-55097.2 uAuGAucGuuucuuuGAGAdTsdT UCUcAAAGAAACGAUcAuAdTsdT 26 27 973-991 AD-55103.2 uGAccAcAccuAucGAGuudTsdT AACUCGAuAGGUGUGGUcAdTsdT 28 29 975-993 AD-55107.2 AccAcAccuAucGAGuuuudTsdT AAAACUCGAuAGGUGUGGUdTsdT 30 31 1029-1047 AD-55112.2 uGGcAGAuGAcuAuucAGAdTsdT UCUGAAuAGUcAUCUGCcAdTsdT 32 33 1077-1095 AD-55074.2 ucuGGuGcAGuAAuGAcuAdTsdT uAGUcAUuACUGcACcAGAdTsdT 34 35 1124-1142 AD-55080.2 uGuGGGGcAGuuAuGGAcAdTsdT UGUCcAuAACUGCCCcAcAdTsdT 36 37 1137-1155 AD-55086.2 uGGAcAcuuuGAAAcAAcAdTsdT UGUUGUUUcAAAGUGUCcAdTsdT 38 39 1182-1200 AD-55098.2 AuAuuucuGGAAcuAGuAAdTsdT UuACuAGUUCcAGAAAuAUdTsdT 40 41 1184-1202 AD-55104.2 AuuucuGGAAcuAGuAAAudTsdT AUUuACuAGUUCcAGAAAUdTsdT 42 43 1185-1203 AD-55108.2 uuucuGGAAcuAGuAAAuudTsdT AAUUuACuAGUUCcAGAAAdTsdT 44 45 1188-1206 AD-55113.2 cuGGAAcuAGuAAAuuccAdTsdT UGGAAUUuACuAGUUCcAGdTsdT 46 47 1325-1343 AD-55075.2 uGuGAGAuuuAcucuGAuudTsdT AAUcAGAGuAAAUCUcAcAdTsdT 48 49 1364-1382 AD-55081.2 AuccAAGGGAuucGAAAcAdTsdT UGUUUCGAAUCCCUUGGAUdTsdT 50 51 1382-1400 AD-55087.2 AGccGAGuGccAAAGuAcAdTsdT UGuACUUUGGcACUCGGCUdTsdT 52 53 1478-1496 AD-55093.2 uuuGAAAcuGuccAuucAAdTsdT UUGAAUGGAcAGUUUcAAAdTsdT 54 55 1531-1549 AD-55099.2 uGAuGuGGcccAuGAGuuudTsdT AAACUcAUGGGCcAcAUcAdTsdT 56 57 1631-1649 AD-53573.3 GucAuGccAAAAAuGGAcAdTsdT UGUCcAUUUUUGGcAUGACdTsdT 58 59 1637-1655 AD-55109.2 ccAAAAAuGGAcAucAuuudTsdT AAAUGAUGUCcAUUUUUGGdTsdT 60 61 1706-1724 AD-55114.2 AcGAGuucucuGAuuGAcAdTsdT UGUcAAUcAGAGAACUCGUdTsdT 62 63 1962-1980 AD-55076.2 AAGucuGuGAuGAAcuAAudTsdT AUuAGUUcAUcAcAGACUUdTsdT 64 65 1967-1985 AD-55082.2 uGuGAuGAAcuAAuGAGcAdTsdT UGCUcAUuAGUUcAUcAcAdTsdT 66 67 1977-1995 AD-55088.2 uAAuGAGcAGAcAuAAcAudTsdT AUGUuAUGUCUGCUcAUuAdTsdT 68 69 2189-2207 AD-55094.2 uuuGAAGuGAuGAGuGAAAdTsdT UUUcACUcAUcACUUcAAAdTsdT 70 71 2227-2245 AD-55100.2 AGGcuuGAGcAAGuuGGuAdTsdT uACcAACUUGCUcAAGCCUdTsdT 72 73 2313-2331 AD-55105.2 ucuucAGAGuuGucuuuAudTsdT AuAAAGAcAACUCUGAAGAdTsdT 74 75 2317-2335 AD-55110.2 cAGAGuuGucuuuAuAuGudTsdT AcAuAuAAAGAcAACUCUGdTsdT 76 77 2319-2337 AD-55115.2 GAGuuGucuuuAuAuGuGAdTsdT UcAcAuAuAAAGAcAACUCdTsdT 78 79 2320-2338 AD-55077.2 AGuuGucuuuAuAuGuGAAdTsdT UUcAcAuAuAAAGAcAACUdTsdT 80 81 2344-2362 AD-55083.2 uuAuAuuAAAuuuuAAucudTsdT AGAUuAAAAUUuAAuAuAAdTsdT 82 83 2352-2370 AD-55089.2 AAuuuuAAucuAuAGuAAAdTsdT UUuACuAuAGAUuAAAAUUdTsdT 84 85 2353-2371 AD-55095.2 AuuuuAAucuAuAGuAAAAdTsdT UUUuACuAuAGAUuAAAAUdTsdT 86 87 2376-2394 AD-55101.2 AGuccuGGAAAuAAAuucudTsdT AGAAUUuAUUUCcAGGACUdTsdT 88 89 358-376 AD-53511.1 cuGcccAuucuuAucccGAdTsdT UCGGGAuAAGAAUGGGcAGdTsdT 90 91 789-807 AD-53512.1 ccAGuGuGGuuAGuGuGAAdTsdT UUcAcACuAACcAcACUGGdTsdT 92 93 1076-1094 AD-53513.1 GucuGGuGcAGuAAuGAcudTsdT AGUcAUuACUGcACcAGACdTsdT 94 95 1253-1271 AD-53514.1 GcAcucuuGuuuuccucGudTsdT ACGAGGAAAAcAAGAGUGCdTsdT 96 97 1544-1562 AD-53515.1 GAGuuuGGAGcAAucAccudTsdT AGGUGAUUGCUCcAAACUCdTsdT 98 99 2228-2246 AD-53516.1 GGcuuGAGcAAGuuGGuAudTsdT AuACcAACUUGCUcAAGCCdTsdT 100 101 404-422 AD-53517.1 GGcAAAucucuGuuGuucudTsdT AGAAcAAcAGAGAUUUGCCdTsdT 102 103 404-422 AD-53517.1 GGcAAAucucuGuuGuucudTsdT AGAAcAAcAGAGAUUUGCCdTsdT 104 105 866-884 AD-53518.1 cAAAGAccAGAAAGAGuGudTsdT AcACUCUUUCUGGUCUUUGdTsdT 106 107 1080-1098 AD-53519.1 GGuGcAGuAAuGAcuAccudTsdT AGGuAGUcAUuACUGcACCdTsdT 108 109 1258-1276 AD-53520.1 cuuGuuuuccucGuGcuuudTsdT AAAGcACGAGGAAAAcAAGdTsdT 110 111 1616-1634 AD-53521.1 GGGGAucGGGAuGGAGucAdTsdT UGACUCcAUCCCGAUCCCCdTsdT 112 113 2230-2248 AD-53522.1 cuuGAGcAAGuuGGuAucudTsdT AGAuACcAACUUGCUcAAGdTsdT 114 115 436-454 AD-53523.1 ccccAAGAuGAuGGAAGuudTsdT AACUUCcAUcAUCUUGGGGdTsdT 116 117 436-454 AD-53523.1 ccccAAGAuGAuGGAAGuudTsdT AACUUCcAUcAUCUUGGGGdTsdT 118 119 885-903 AD-53524.1 cucAucuucuucAAGAuAAdTsdT UuAUCUUGAAGAAGAUGAGdTsdT 120 121 1127-1145 AD-53525.1 GGGGcAGuuAuGGAcAcuudTsdT AAGUGUCcAuAACUGCCCCdTsdT 122 123 1315-1333 AD-53526.1 GAuGccAGGcuGuGAGAuudTsdT AAUCUcAcAGCCUGGcAUCdTsdT 124 125 1870-1888 AD-53527.1 GAGAcAGAuGcuAAuGGAudTsdT AUCcAUuAGcAUCUGUCUCdTsdT 126 127 2286-2304 AD-53528.1 ccccAGGccAuuAucAuAudTsdT AuAUGAuAAUGGCCUGGGGdTsdT 128 129 489-507 AD-53529.1 cAGcAGuAcAcuAccAAcAdTsdT UGUUGGuAGUGuACUGCUGdTsdT 130 131 489-507 AD-53529.1 cAGcAGuAcAcuAccAAcAdTsdT UGUUGGuAGUGuACUGCUGdTsdT 132 133 915-933 AD-53530.1 cuGuuuccAcuuuucAGuAdTsdT uACUGAAAAGUGGAAAcAGdTsdT 134 135 1138-1156 AD-53531.1 GGAcAcuuuGAAAcAAcAudTsdT AUGUUGUUUcAAAGUGUCCdTsdT 136 137 1324-1342 AD-53532.1 cuGuGAGAuuuAcucuGAudTsdT AUcAGAGuAAAUCUcAcAGdTsdT 138 139 1927-1945 AD-53533.1 cccuGuGcGGGuuGcAGAudTsdT AUCUGcAACCCGcAcAGGGdTsdT 140 141 2312-2330 AD-53534.1 GucuucAGAGuuGucuuuAdTsdT uAAAGAcAACUCUGAAGACdTsdT 142 143 646-664 AD-53535.1 cAcuGcAAGcAAAuGcccudTsdT AGGGcAUUUGCUUGcAGUGdTsdT 144 145 922-940 AD-53536.1 cAcuuuucAGuAuGAucGudTsdT ACGAUcAuACUGAAAAGUGdTsdT 146 147 1163-1181 AD-53537.1 GGGGcAGGuGGuAcuAGAAdTsdT UUCuAGuACcACCUGCCCCdTsdT 148 149 1347-1365 AD-53538.1 GGAAccAuGccuccAuGAudTsdT AUcAUGGAGGcAUGGUUCCdTsdT 150 151 1964-1982 AD-53539.1 GucuGuGAuGAAcuAAuGAdTsdT UcAUuAGUUcAUcAcAGACdTsdT 152 153 2321-2339 AD-53540.1 GuuGucuuuAuAuGuGAAudTsdT AUUcAcAuAuAAAGAcAACdTsdT 154 155 671-689 AD-53541.1 GcAGcAcAGAuGAAucAGAdTsdT UCUGAUUcAUCUGUGCUGCdTsdT 156 157 924-942 AD-53542.1 cuuuucAGuAuGAucGuuudTsdT AAACGAUcAuACUGAAAAGdTsdT 158 159 1164-1182 AD-53543.1 GGGcAGGuGGuAcuAGAAAdTsdT UUUCuAGuACcACCUGCCCdTsdT 160 161 1460-1478 AD-53544.1 GuccccAAGAuuGuGGcAudTsdT AUGCcAcAAUCUUGGGGACdTsdT 162 163 1976-1994 AD-53545.1 cuAAuGAGcAGAcAuAAcAdTsdT UGUuAUGUCUGCUcAUuAGdTsdT 164 165 786-804 AD-53546.1 GccccAGuGuGGuuAGuGudTsdT AcACuAACcAcACUGGGGCdTsdT 166 167 935-953 AD-53547.1 GAucGuuucuuuGAGAAAAdTsdT UUUUCUcAAAGAAACGAUCdTsdT 168 169 1165-1183 AD-53548.1 GGcAGGuGGuAcuAGAAAudTsdT AUUUCuAGuACcACCUGCCdTsdT 170 171 1530-1548 AD-53549.1 GuGAuGuGGcccAuGAGuudTsdT AACUcAUGGGCcAcAUcACdTsdT 172 173 2003-2021 AD-53550.1 cAAGcAAucAAuuAcccuAdTsdT uAGGGuAAUUGAUUGCUUGdTsdT 174 175 788-806 AD-53551.1 cccAGuGuGGuuAGuGuGAdTsdT UcAcACuAACcAcACUGGGdTsdT 176 177 974-992 AD-53552.1 GAccAcAccuAucGAGuuudTsdT AAACUCGAuAGGUGUGGUCdTsdT 178 179 1191-1209 AD-53553.1 GAAcuAGuAAAuuccAuGudTsdT AcAUGGAAUUuACuAGUUCdTsdT 180 181 1541-1559 AD-53554.1 cAuGAGuuuGGAGcAAucAdTsdT UGAUUGCUCcAAACUcAUGdTsdT 182 183 2075-2093 AD-53555.1 ccccAGAuGAuGAAcuAcudTsdT AGuAGUUcAUcAUCUGGGGdTsdT 184 185 360-378 AD-53561.1 GcccAuucuuAucccGAGudTsdT ACUCGGGAuAAGAAUGGGCdTsdT 186 187 1356-1374 AD-53567.1 ccuccAuGAuccAAGGGAudTsdT AUCCCUUGGAUcAUGGAGGdTsdT 188 189 1631-1649 AD-53573.1 GucAuGccAAAAAuGGAcAdTsdT UGUCcAUUUUUGGcAUGACdTsdT 190 191 1634-1652 AD-53579.1 AuGccAAAAAuGGAcAucAdTsdT UGAUGUCcAUUUUUGGcAUdTsdT

TABLE 3 Human ALAS1 Unmodified Single Strands and Duplex Sequences SEQ ID SEQ ID NO: Position on NO: (anti- transcript Duplex Sense Sequence Antisense Sequence (sense) sense) NM_000688.4 Name (5′-3′) (5'-3') 192 193 522-540 AD-55078.2 CUCCGGCCAGUGAGAAAGA UCUUUCUCACUGGCCGGAG 194 195 669-687 AD-55084.2 UGGCAGCACAGAUGAAUCA UGAUUCAUCUGUGCUGCCA 196 197 790-808 AD-55090.2 CAGUGUGGUUAGUGUGAAA UUUCACACUAACCACACUG 198 199 853-871 AD-55096.2 CAUCAUGCAAAAGCAAAGA UCUUUGCUUUUGCAUGAUG 200 201 876-894 AD-55102.2 AAAGAGUGUCUCAUCUUCU AGAAGAUGAGACACUCUUU 202 203 877-895 AD-55106.2 AAGAGUGUCUCAUCUUCUU AAGAAGAUGAGACACUCUU 204 205 914-932 AD-55111.2 UCUGUUUCCACUUUUCAGU ACUGAAAAGUGGAAACAGA 206 207 923-941 AD-55073.2 ACUUUUCAGUAUGAUCGUU AACGAUCAUACUGAAAAGU 208 209 926-944 AD-55079.2 UUUCAGUAUGAUCGUUUCU AGAAACGAUCAUACUGAAA 210 211 927-945 AD-55085.2 UUCAGUAUGAUCGUUUCUU AAGAAACGAUCAUACUGAA 212 213 928-946 AD-55091.2 UCAGUAUGAUCGUUUCUUU AAAGAAACGAUCAUACUGA 214 215 932-950 AD-55097.2 UAUGAUCGUUUCUUUGAGA UCUCAAAGAAACGAUCAUA 216 217 973-991 AD-55103.2 UGACCACACCUAUCGAGUU AACUCGAUAGGUGUGGUCA 218 219 975-993 AD-55107.2 ACCACACCUAUCGAGUUUU AAAACUCGAUAGGUGUGGU 220 221 1029-1047 AD-55112.2 UGGCAGAUGACUAUUCAGA UCUGAAUAGUCAUCUGCCA 222 223 1077-1095 AD-55074.2 UCUGGUGCAGUAAUGACUA UAGUCAUUACUGCACCAGA 224 225 1124-1142 AD-55080.2 UGUGGGGCAGUUAUGGACA UGUCCAUAACUGCCCCACA 226 227 1137-1155 AD-55086.2 UGGACACUUUGAAACAACA UGUUGUUUCAAAGUGUCCA 228 229 1182-1200 AD-55098.2 AUAUUUCUGGAACUAGUAA UUACUAGUUCCAGAAAUAU 230 231 1184-1202 AD-55104.2 AUUUCUGGAACUAGUAAAU AUUUACUAGUUCCAGAAAU 232 233 1185-1203 AD-55108.2 UUUCUGGAACUAGUAAAUU AAUUUACUAGUUCCAGAAA 234 235 1188-1206 AD-55113.2 CUGGAACUAGUAAAUUCCA UGGAAUUUACUAGUUCCAG 236 237 1325-1343 AD-55075.2 UGUGAGAUUUACUCUGAUU AAUCAGAGUAAAUCUCACA 238 239 1364-1382 AD-55081.2 AUCCAAGGGAUUCGAAACA UGUUUCGAAUCCCUUGGAU 240 241 1382-1400 AD-55087.2 AGCCGAGUGCCAAAGUACA UGUACUUUGGCACUCGGCU 242 243 1478-1496 AD-55093.2 UUUGAAACUGUCCAUUCAA UUGAAUGGACAGUUUCAAA 244 245 1531-1549 AD-55099.2 UGAUGUGGCCCAUGAGUUU AAACUCAUGGGCCACAUCA 246 247 1631-1649 AD-53573.3 GUCAUGCCAAAAAUGGACA UGUCCAUUUUUGGCAUGAC 248 249 1637-1655 AD-55109.2 CCAAAAAUGGACAUCAUUU AAAUGAUGUCCAUUUUUGG 250 251 1706-1724 AD-55114.2 ACGAGUUCUCUGAUUGACA UGUCAAUCAGAGAACUCGU 252 253 1962-1980 AD-55076.2 AAGUCUGUGAUGAACUAAU AUUAGUUCAUCACAGACUU 254 255 1967-1985 AD-55082.2 UGUGAUGAACUAAUGAGCA UGCUCAUUAGUUCAUCACA 256 257 1977-1995 AD-55088.2 UAAUGAGCAGACAUAACAU AUGUUAUGUCUGCUCAUUA 258 259 2189-2207 AD-55094.2 UUUGAAGUGAUGAGUGAAA UUUCACUCAUCACUUCAAA 260 261 2227-2245 AD-55100.2 AGGCUUGAGCAAGUUGGUA UACCAACUUGCUCAAGCCU 262 263 2313-2331 AD-55105.2 UCUUCAGAGUUGUCUUUAU AUAAAGACAACUCUGAAGA 264 265 2317-2335 AD-55110.2 CAGAGUUGUCUUUAUAUGU ACAUAUAAAGACAACUCUG 266 267 2319-2337 AD-55115.2 GAGUUGUCUUUAUAUGUGA UCACAUAUAAAGACAACUC 268 269 2320-2338 AD-55077.2 AGUUGUCUUUAUAUGUGAA UUCACAUAUAAAGACAACU 270 271 2344-2362 AD-55083.2 UUAUAUUAAAUUUUAAUCU AGAUUAAAAUUUAAUAUAA 272 273 2352-2370 AD-55089.2 AAUUUUAAUCUAUAGUAAA UUUACUAUAGAUUAAAAUU 274 275 2353-2371 AD-55095.2 AUUUUAAUCUAUAGUAAAA UUUUACUAUAGAUUAAAAU 276 277 2376-2394 AD-55101.2 AGUCCUGGAAAUAAAUUCU AGAAUUUAUUUCCAGGACU 278 279 358-376 AD-53511.1 CUGCCCAUUCUUAUCCCGA UCGGGAUAAGAAUGGGCAG 280 281 789-807 AD-53512.1 CCAGUGUGGUUAGUGUGAA UUCACACUAACCACACUGG 282 283 1076-1094 AD-53513.1 GUCUGGUGCAGUAAUGACU AGUCAUUACUGCACCAGAC 284 285 1253-1271 AD-53514.1 GCACUCUUGUUUUCCUCGU ACGAGGAAAACAAGAGUGC 286 287 1544-1562 AD-53515.1 GAGUUUGGAGCAAUCACCU AGGUGAUUGCUCCAAACUC 288 289 2228-2246 AD-53516.1 GGCUUGAGCAAGUUGGUAU AUACCAACUUGCUCAAGCC 290 291 404-422 AD-53517.1 GGCAAAUCUCUGUUGUUCU AGAACAACAGAGAUUUGCC 292 293 404-422 AD-53517.1 GGCAAAUCUCUGUUGUUCU AGAACAACAGAGAUUUGCC 294 295 866-884 AD-53518.1 CAAAGACCAGAAAGAGUGU ACACUCUUUCUGGUCUUUG 296 297 1080-1098 AD-53519.1 GGUGCAGUAAUGACUACCU AGGUAGUCAUUACUGCACC 298 299 1258-1276 AD-53520.1 CUUGUUUUCCUCGUGCUUU AAAGCACGAGGAAAACAAG 300 301 1616-1634 AD-53521.1 GGGGAUCGGGAUGGAGUCA UGACUCCAUCCCGAUCCCC 302 303 2230-2248 AD-53522.1 CUUGAGCAAGUUGGUAUCU AGAUACCAACUUGCUCAAG 304 305 436-454 AD-53523.1 CCCCAAGAUGAUGGAAGUU AACUUCCAUCAUCUUGGGG 306 307 436-454 AD-53523.1 CCCCAAGAUGAUGGAAGUU AACUUCCAUCAUCUUGGGG 308 309 885-903 AD-53524.1 CUCAUCUUCUUCAAGAUAA UUAUCUUGAAGAAGAUGAG 310 311 1127-1145 AD-53525.1 GGGGCAGUUAUGGACACUU AAGUGUCCAUAACUGCCCC 312 313 1315-1333 AD-53526.1 GAUGCCAGGCUGUGAGAUU AAUCUCACAGCCUGGCAUC 314 315 1870-1888 AD-53527.1 GAGACAGAUGCUAAUGGAU AUCCAUUAGCAUCUGUCUC 316 317 2286-2304 AD-53528.1 CCCCAGGCCAUUAUCAUAU AUAUGAUAAUGGCCUGGGG 318 319 489-507 AD-53529.1 CAGCAGUACACUACCAACA UGUUGGUAGUGUACUGCUG 320 321 489-507 AD-53529.1 CAGCAGUACACUACCAACA UGUUGGUAGUGUACUGCUG 322 323 915-933 AD-53530.1 CUGUUUCCACUUUUCAGUA UACUGAAAAGUGGAAACAG 324 325 1138-1156 AD-53531.1 GGACACUUUGAAACAACAU AUGUUGUUUCAAAGUGUCC 326 327 1324-1342 AD-53532.1 CUGUGAGAUUUACUCUGAU AUCAGAGUAAAUCUCACAG 328 329 1927-1945 AD-53533.1 CCCUGUGCGGGUUGCAGAU AUCUGCAACCCGCACAGGG 330 331 2312-2330 AD-53534.1 GUCUUCAGAGUUGUCUUUA UAAAGACAACUCUGAAGAC 332 333 646-664 AD-53535.1 CACUGCAAGCAAAUGCCCU AGGGCAUUUGCUUGCAGUG 334 335 922-940 AD-53536.1 CACUUUUCAGUAUGAUCGU ACGAUCAUACUGAAAAGUG 336 337 1163-1181 AD-53537.1 GGGGCAGGUGGUACUAGAA UUCUAGUACCACCUGCCCC 338 339 1347-1365 AD-53538.1 GGAACCAUGCCUCCAUGAU AUCAUGGAGGCAUGGUUCC 340 341 1964-1982 AD-53539.1 GUCUGUGAUGAACUAAUGA UCAUUAGUUCAUCACAGAC 342 343 2321-2339 AD-53540.1 GUUGUCUUUAUAUGUGAAU AUUCACAUAUAAAGACAAC 344 345 671-689 AD-53541.1 GCAGCACAGAUGAAUCAGA UCUGAUUCAUCUGUGCUGC 346 347 924-942 AD-53542.1 CUUUUCAGUAUGAUCGUUU AAACGAUCAUACUGAAAAG 348 349 1164-1182 AD-53543.1 GGGCAGGUGGUACUAGAAA UUUCUAGUACCACCUGCCC 350 351 1460-1478 AD-53544.1 GUCCCCAAGAUUGUGGCAU AUGCCACAAUCUUGGGGAC 352 353 1976-1994 AD-53545.1 CUAAUGAGCAGACAUAACA UGUUAUGUCUGCUCAUUAG 354 355 786-804 AD-53546.1 GCCCCAGUGUGGUUAGUGU ACACUAACCACACUGGGGC 356 357 935-953 AD-53547.1 GAUCGUUUCUUUGAGAAAA UUUUCUCAAAGAAACGAUC 358 359 1165-1183 AD-53548.1 GGCAGGUGGUACUAGAAAU AUUUCUAGUACCACCUGCC 360 361 1530-1548 AD-53549.1 GUGAUGUGGCCCAUGAGUU AACUCAUGGGCCACAUCAC 362 363 2003-2021 AD-53550.1 CAAGCAAUCAAUUACCCUA UAGGGUAAUUGAUUGCUUG 364 365 788-806 AD-53551.1 CCCAGUGUGGUUAGUGUGA UCACACUAACCACACUGGG 366 367 974-992 AD-53552.1 GACCACACCUAUCGAGUUU AAACUCGAUAGGUGUGGUC 368 369 1191-1209 AD-53553.1 GAACUAGUAAAUUCCAUGU ACAUGGAAUUUACUAGUUC 370 371 1541-1559 AD-53554.1 CAUGAGUUUGGAGCAAUCA UGAUUGCUCCAAACUCAUG 372 373 2075-2093 AD-53555.1 CCCCAGAUGAUGAACUACU AGUAGUUCAUCAUCUGGGG 374 375 360-378 AD-53561.1 GCCCAUUCUUAUCCCGAGU ACUCGGGAUAAGAAUGGGC 376 377 1356-1374 AD-53567.1 CCUCCAUGAUCCAAGGGAU AUCCCUUGGAUCAUGGAGG 378 379 1631-1649 AD-53573.1 GUCAUGCCAAAAAUGGACA UGUCCAUUUUUGGCAUGAC 380 381 1634-1652 AD-53579.1 AUGCCAAAAAUGGACAUCA UGAUGUCCAUUUUUGGCAU

Example 3. In Vitro Screening of ALAS1 siRNA Duplexes for ALAS1 Knockdown Activity

ALAS1 siRNA duplexes were screened for the ability to knockdown ALAS1 expression in vitro.

In Vitro Screening

Cell Culture and Transfections

Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in MEM (ATCC) supplemented with 10% FBS, before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media containing ˜2×10⁴ Hep3B cells were then added to the siRNA mixture. Cells were incubated for either 24 or 120 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentration and dose response experiments were done at 10, 1.67, 0.27, 0.046, 0.0077, 0.0013, 0.00021, 0.00004 nM final duplex concentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12)

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing supernatant, magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixed for 1 minute. Beads were captured again and supernatant removed. Beads were then washed with 150 μl Wash Buffer B, captured and supernatant was removed. Beads were next washed with 150 μl Elution Buffer, captured and supernatant removed. Beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H₂O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.

Real Time PCR

2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl ALAS1 TaqMan probe (Applied Biosystems cat #Hs00167441_m1) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plates (Roche cat #04887301001). Real time PCR was done in a Roche LC480 Real Time PCR system (Roche) using the ΔΔCt(RQ) assay. Each duplex was tested in two independent transfections with two biological replicates each, and each transfection was assayed in duplicate, unless otherwise noted in the summary tables.

To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC50s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 or naïve cells over the same dose range, or to its own lowest dose.

In Vitro Knockdown of Endogenous ALAS1 Expression by ALAS1 siRNA Duplexes

Table 4 illustrates the knockdown of ALAS1 in Hep3B cells by ALAS1 modified siRNA duplexes (See Table 2). Silencing is expressed as the fraction RNA message remaining relative to the negative (luciferase) control siRNA AD-1955. Data were generated as described above following transfection of 10 nM or 0.1 nM of each siRNA. qPCR was run using the ALAS1 TaqMan probe Hs00167441_m1.

TABLE 4 ALAS1 expression in Hep3B cells following transfection with ALAS1 siRNA Duplex ID 10 nM Avg 0.1 nM Avg 10 nM STDEV 0.1 nM STDEV AD-55078.2 0.7 0.87 0.001 0.089 AD-55084.2 0.08 0.3 0 0.04 AD-55090.2 0.06 0.08 0.002 0.003 AD-55096.2 0.61 0.92 0.171 0.34 AD-55102.2 0.63 0.62 0.005 0.069 AD-55106.2 0.07 0.08 0.004 0.027 AD-55111.2 0.06 0.23 0.013 0.062 AD-55073.2 0.21 0.4 0.018 0.061 AD-55079.2 0.17 0.43 0.033 0.089 AD-55085.2 0.13 0.21 0.011 0.019 AD-55091.2 0.27 0.55 0.033 0.009 AD-55097.2 0.31 0.38 0.051 0.059 AD-55103.2 0.05 0.11 0.017 0.006 AD-55107.2 0.12 0.24 0.007 0.008 AD-55112.2 0.15 0.2 0.036 0.025 AD-55074.2 0.16 0.45 0.008 0.002 AD-55080.2 0.79 0.99 0.095 0.304 AD-55086.2 0.09 0.22 0.005 0.035 AD-55098.2 0.25 0.51 0.03 0.07 AD-55104.2 0.06 0.1 0.017 0.001 AD-55108.2 0.47 0.65 0.03 0.015 AD-55113.2 0.38 0.62 0.068 0.039 AD-55075.2 0.12 0.28 0.007 0.051 AD-55081.2 0.21 0.51 0.036 0.066 AD-55087.2 0.1 0.19 0.017 0.02 AD-55093.2 0.24 0.56 0.029 0.053 AD-55099.2 0.05 0.18 0.001 0.038 AD-53573.3 0.67 1.07 0.16 0.153 AD-55109.2 0.07 0.23 0.006 0.052 AD-55114.2 0.08 0.16 0.004 0.017 AD-55076.2 0.05 0.14 0.007 0.035 AD-55082.2 0.08 0.3 0.019 0.016 AD-55088.2 0.06 0.12 0.008 0.02 AD-55094.2 0.06 0.18 0.005 0.023 AD-55100.2 0.45 0.83 0.02 0.05 AD-55105.2 0.02 0.05 0.005 0.004 AD-55110.2 0.15 0.19 0.031 0.016 AD-55115.2 0.35 0.58 0.045 0.052 AD-55077.2 0.14 0.14 0.006 0.019 AD-55083.2 0.56 0.98 0.24 0.188 AD-55089.2 0.62 0.79 0.036 0.094 AD-55095.2 0.59 0.92 0.12 0.079 AD-55101.2 0.71 0.97 0.074 0.097 AD-1955 1.00 1.01 0.03 0.04 AD-53511.1 0.84 1.08 0.028 0.0515 AD-53512.1 0.15 0.65 0.062 0.023 AD-53513.1 0.34 0.86 0.055 0.011 AD-53514.1 0.12 0.61 0.003 0.008 AD-53515.1 0.25 0.66 0.005 0.004 AD-53516.1 1.05 1.02 0.032 0.011 AD-53517.1 0.145 0.725 0.025 0.0155 AD-53518.1 0.72 0.85 0.045 0.028 AD-53519.1 0.18 0.66 0.061 0.004 AD-53520.1 0.18 0.9 0.041 0.001 AD-53521.1 0.97 1.07 0.01 0.003 AD-53522.1 0.87 1.1 0.065 0.112 AD-53523.1 0.48 0.96 0.0305 0.0255 AD-53524.1 0.11 0.66 0.02 0.006 AD-53525.1 0.71 1.03 0.016 0.01 AD-53526.1 0.23 0.85 0.075 0.01 AD-53527.1 0.25 0.83 0.015 0.017 AD-53528.1 0.44 0.93 0.037 0.006 AD-53529.1 0.185 0.73 0.015 0.014 AD-53530.1 0.1 0.62 0.02 0.003 AD-53531.1 0.48 0.93 0.019 0.045 AD-53532.1 0.06 0.17 0 0.003 AD-53533.1 0.36 0.93 0.025 0.034 AD-53534.1 0.1 0.36 0.014 0.012 AD-53535.1 0.58 1.05 0.036 0.071 AD-53536.1 0.12 0.45 0.009 0.026 AD-53537.1 0.73 0.96 0.101 0.015 AD-53538.1 0.74 1.07 0 0.046 AD-53539.1 0.52 0.97 0.057 0.032 AD-53540.1 0.1 0.47 0.017 0.012 AD-53541.1 0.11 0.29 0.026 0.015 AD-53542.1 0.08 0.23 0.008 0.006 AD-53543.1 0.62 1.01 0.027 0.014 AD-53544.1 0.8 1.04 0.002 0.001 AD-53545.1 0.17 0.73 0.007 0.007 AD-53546.1 0.27 0.93 0.058 0.019 AD-53547.1 0.12 0.28 0.008 0.01 AD-53548.1 0.1 0.34 0.022 0.002 AD-53549.1 0.8 1.04 0.011 0.026 AD-53550.1 0.05 0.54 0.02 0.003 AD-53551.1 0.96 1.16 0.029 0.044 AD-53552.1 0.13 0.5 0.002 0.009 AD-53553.1 0.92 1.1 0.027 0.02 AD-53554.1 0.76 0.67 0.005 0.004 AD-53555.1 0.11 0.53 0.009 0.007 AD-53561.1 0.72 0.94 0.014 0.001 AD-53567.1 0.16 0.66 0.019 0.003 AD-53573.1 1.06 1.10 0.019 0.037 AD-53579.1 0.19 0.76 0.036 0.019 IC₅₀s of Select ALAS1 siRNA Duplexes in In Vitro Screen

Table 5 illustrates the IC₅₀s of select ALAS1 siRNA duplexes determined from the knockdown of endogenously expressed ALAS1 in the Hep3B cell line, by ALAS1 modified siRNA duplexes (see Table 2). Data were generated as described above, at 24 or 120 hours following transfection of each siRNA duplex. Silencing of ALAS1 is expressed as the fraction mRNA message remaining relative to the siRNA AD-1955, a non-targeting siRNA that was used as a negative control. Data from replicate transfection experiments were used to fit a single line to determine the IC₅₀. Several of the duplexes (e.g., AD-53541.1, AD-53542.1, and AD-53547.1) had an IC₅₀ as low as about 0.03 nM at 24 hours. Numerous duplexes had an IC₅₀ of less than 0.1 nM (e.g., AD-53534.1, AD-53536.1, AD-53540.1, AD-53541.1, AD-53542.1, AD-53547.1, AD-53548.1, AD-53550.1, AD-53552.1) at 24 hours, and some of these also had an IC₅₀ of less than 0.1 nM (e.g., AD-53534.1, AD-53540.1, AD-53541.1, AD-53542.1, AD-53547.1, AD-53552.1) at 120 hours.

TABLE 5 IC_(50S) of select ALAS1 siRNA duplexes normalized to AD-1955 IC₅₀ (nM) DUPLEX ID 24 hrs 120 hrs AD-53534.1 0.045 0.076 AD-53536.1 0.049 0.105 AD-53540.1 0.054 0.077 AD-53541.1 0.032 0.062 AD-53542.1 0.028 0.093 AD-53547.1 0.03 0.062 AD-53548.1 0.044 0.101 AD-53550.1 0.085 0.152 AD-53552.1 0.077 0.063 AD-53567.1 0.219 0.357 AD-53579.1 0.217 0.566

Example 4. In Vivo Silencing Using a Mouse/Rat ALAS1 siRNA Formulated as a LNP

The sequences of the modified duplex AD-53558 are shown in Table 6 below.

TABLE 6 Sequences of ALAS1 siRNA Duplex AD-53558.4 SEQ ID Start SEQ ID NO: Position on NO: (anti- transcript of Duplex Sense Sequence Antisense Sequence (sense) sense) NM_020559.2 Name (5′-3′) (5′-3′) 383 384 1184 AD-53558 cuGuGAAAuuuAcucuGAudTsdT AUcAGAGuAAAUUUcAcAGdTsdT

This duplex was formulated as a LNP11 formulation (see Table 10 above). The LNP-formulated AD-53558 siRNA was tested in in vivo in mice (N=25 animals; 5 animals per group) and rats (N=20 animals; 4 animals per group) and was confirmed to silence ALAS1 mRNA in vivo. The results are shown in FIG. 5 and FIG. 6.

FIG. 5 shows that the siRNA demonstrated a dose-response effect in mice. The expression of mouse ALAS1 (mALAS1) mRNA was reduced by about 78% when the siRNA was administered at 1 mg/kg; mouse ALAS1 mRNA was reduced by about 60% when the siRNA was administered at 0.3 mg/kg; and mouse ALAS1 mRNA was reduced by about 49% when the siRNA was administered at 0.1 mg/kg. These reductions are expressed relative to a PBS control. An AD-1955 LUC control was also employed, as shown in FIG. 5.

Similarly, FIG. 6 shows that the siRNA demonstrated a dose-response effect in rats. The expression of ALAS1 RNA was reduced by about 70% when the when the siRNA was administered at 1 mg/kg; ALAS1 mRNA was reduced by about 62% when the siRNA was administered at 0.3 mg/kg; and ALAS1 mRNA was reduced by about 34% when the siRNA was administered at 0.1 mg/kg.

The durability of silencing was also tested in mice (N=15; 3 animals per timepoint. The results are shown in FIG. 7, which shows that AD-53558 suppressed mALAS1 mRNA by about 80% for at least 9 days. Suppression of at least about 50% persisted for at least 14 days.

Example 5. Efficacy of ALAS1 siRNA in an Animal Model of AIP

The effects of the AD-53558 LNP11 formulation (a mouse/rat ALAS1 siRNA described in the previous example) were investigated in a mouse model of AP. The PBGD knockout is not viable (−/−, 0% activity). Heterozygous PBGD knockout mice (+/−, 50% activity) are available but do not have the full biochemical phenotype and thus do not recapitulate the human disease phenotype. Thus, a mouse model of AIP has been developed that is a compound heterozygote with T1/T2 alleles, including T1 (+/−) promoter disruption and T2 (−/−) splice-site alteration. These mice have been shown to have hepatic residual PBGD activity that is about ˜30% of the wild-type level and normal or slightly elevated baseline plasma ALA and PBG levels. The mice have been found to appear normal early in life and to become slightly slower and ataxic with age. By six months of age, the mice have been documented to develop impaired motor coordination and muscular performance and axonal degeneration on pathological examination. Investigation of the pathology of the mouse model has shown axonal degeneration, impaired motor coordination and muscular performance in older mice. Urinary and plasma ALA and PBG have been found to markedly increase with serial i.p. administration of phenobarbital (see Lindberg et al., (1996), Nature Genetics, 12:195-219 and Lindberg et al., (1999), Journal of Clinical Investigation, 103:1127-34). The mice were rescued by AAV-mediated expression of PBGD in the liver (Yasuda et al. (2010), Molecular Medicine, 1:17-22 and Unzu et al. (2011), Molecular Medicine, 2:243-50).

On day 1, the mice were administered 1 mg/kg ALAS1 siRNA (n=5) or LUC AD-1955 control (n=3) by i.v. injection. Three phenobarbitol injections were given (1 injection per day on days 2, 3, and 4) to induce hepatic ALAS1 ande the porphyrin precursors, ALA and PBG. Plasma and overnight urine specimens were collected on day 5 and metabolite levels were measured by LC-MS. Metabolite levels were measured in plasma by LC-MS and were also measured in urine. Baseline levels of metabolites were measured prior to the first treatment on day 1. The results are shown in FIGS. 8-12 and in Tables 12 and 13.

FIG. 8 and FIG. 9 show the plasma ALA levels in μM. Baseline ALA levels were low, (n=4), and phenobarbitol treatment induced significant increases in plasma ALA levels in the control LUC siRNA treated animals (n=3). Treatment with ALAS1 siRNA inhibited the induction of plasma ALA (n=5), as shown in FIG. 8. The ALAS1 siRNA was consistently effective in blocking the induction of plasma ALA in each of the individual animals studied (see FIG. 9). These results indicate that ALAS1 siRNA treatment was effective in preventing the increases in plasma ALA associated with the phenobarbital-induced acute attacks in this AIP animal model.

FIG. 10 and FIG. 11 show the plasma PBG levels in μM. Baseline PBG levels were low (n=4), and phenobarbitol treatment induced significant increases in plasma PBG levels in the control LUC siRNA treated animals (n=3) Treatment with ALAS1 siRNA inhibited the induction of plasma PBG (n=5), as shown in FIG. 10. The ALAS1 siRNA was consistently effective in blocking the induction of plasma PBG in each of the individual animals studied (see FIG. 11). These results indicate that ALAS1 siRNA treatment was effective in preventing the increases in plasma PBG associated with the phenobarbital-induced acute attacks in this AP animal model.

Tables 12 and 13 shows urine ALA and PBG levels at baseline and after phenobarbitol treatment in LUC siRNA (n=2) control (CTR, which refers to a PBS buffer treated animal, n=1) and ALAS1 siRNA (n=5) treated animals.

TABLE 12 Urine data from individual animals showing prevention of induced acute attack ALA PBG ALA PBG (micro (micro Creatinine (microM/mg (microM/mg Mouse ID M/I) M/L) (mg/dl) creatinine) creatinine) siRNA PB Ha-17-4-6 29.7 7.9 Baseline − Ha-19-5-4/2 15.7 5.1 Baseline − Ha-20-39- 28.6 6.7 Baseline − 4/3 Ha-20-38-4 21.4 4.7 Baseline − Ha-21-33-4 934.92 483.71 0.4205 222.33 115.03 Luc + Ha-21-36-9 944.08 563.53 0.5055 186.76 111.48 Luc + Ha-21-18-8 32.88 8.69 0.133 24.72 6.53 ALAS1; + 1 mg/kg Ha-21-33-7 83.07 23.28 0.426 19.50 5.46 ALAS1; + 1 mg/kg Ha-21-34-5 59.15 18.41 0.263 22.49 7.00 ALAS1; + 1 mg/kg PB stands for phenobarbitol. A ″+″ indicates that phenobarbitol was administered.

TABLE 13 Average Urine Data Mean ALA Mean PBG (microM/mg creatinine) (microM/mg creatinine) 23.8 6.1 AIP Baseline 204.55 113.26 Luc-siRNA 22.24 6.33 ALAS1-siRNA

Phenobarbitol treatment induced strong increases (˜25-30 fold increases) in urine ALA (˜9-fold over baseline levels) and PBG (˜19-fold over baseline levels) in the LUC siRNA treated mice, control, whereas such increases were not observed in the ALAS1 siRNA treated animals. Thus, ALAS1 siRNA blocked phenobarbitol-induced increases in urinary ALA and PBG. These results are consistent with the plasma measurements and show that ALAS1 siRNA treatment was effective in preventing increases in urinary metabolites (ALA and PBG) associated with the phenobarbital-induced acute attacks in this AIP animal model.

In further experiments (FIG. 12), it was found that phenobarbitol treatment induced large increases (˜25 fold) in ALAS1 mRNA expression in the liver of the mouse model. Administration of ALAS1 siRNA completely blocked this ALAS1 mRNA induction. These results provide further evidence that ALAS1 siRNA is effective in an animal model of AIP.

Collectively, the results provided in this Example show that ALAS1 siRNA was effective in treating acute attacks in an animal model of the acute hepatic porphyria AIP. Multiple outcome measures support this conclusion, including plasma ALA levels, plasma PBG levels, urine ALA levels, urine PBG levels, and liver ALAS1 mRNA expression levels.

Example 6. In Vivo Silencing Using GalNAc-Conjugated Mouse ALAS1 siRNA

The experiments described in this example investigated the in vivo efficacy of three GalNAc-conjugated siRNAs (see Table 7). These siRNAs were designed and produced with methods such as those described in Example 2.

TABLE 7 Sequences AD-57929 Position SEQ Position of SEQ ID of sense antisense ID NO: seq. on seq. on NO: (anti- transcript Duplex Sense Sequence Antisense Sequence transcript (sense) sense) NM_ 020559.2 Name (5′-3′) (5′-3′) NM_020559.2 385 386 775-795 AD- AfaGfuCfuGfuUfUfCf uUfgAfaAfaGfuGfgaa 773-795 56211 cAfcUfuUfuCfaAfL96 AfcAfgAfcUfusUfsg 387 388 2168-2188 AD- AfcAfuAfgUfaGfCfCf aGfaCfaAfuUfcUfggc 2166-2188 56173 aGfaAfuUfgUfcUfL96 UfaCfuAfuGfusGfsg 389 390 775-795 AD- AfsasGfuCfuGfuUfUf usUfsgAfaAfaGfuGfg 773-795 57929 CfcAfcUfuUfuCfaAfL aaAfcAfgAfcUfususg 96

The mice (n=40; n=4 per experimental condition) were divided into groups that received PBS or doses of 3 mg/kg, 10 mg/kg, or 30 mg/kg of siRNA administered subcutaneously. The level of mALAS1/mGAPDH mRNA, relative to the PBS control, was determined in liver cells at 72 hours post-administration. The results are shown in FIG. 13. There was not a clear dose-response effect for the siRNAs AD-56211 and AD-56173. In contrast, the ALAS1 siRNA AD-57929 showed a dose-response effect in inhibiting mALAS1 expression. These results demonstrate that an ALAS1 GalNAc conjugate was effective in inhibiting expression of ALAS1 mRNA in vivo and showed a dose-response effect.

Example 7. Human siRNAs

Additional human siRNAs were designed and produced as described in Example 2. The top 45 siRNAs were selected based on their predicted efficacy. The sequences of these 45 siRNAs are provided in Table 8.

TABLE 8 Human ALAS1 siRNA Sense and Antisense Sequences SEQ ID Position SEQ ID NO: on NO: (anti- transcript Sense Sequence Antisense Sequence (sense) sense) NM_000688.4 (5′-3′) (5′-3′) 391 392 1635-1657 CAUGCCAAAAAUGGACAUCAU AUGAUGUCCAUUUUUGGCAUGAC 393 394 2352-2374 UAAAUUUUAAUCUAUAGUAAA UUUACUAUAGAUUAAAAUUUAAU 395 396 1324-1346 GGCUGUGAGAUUUACUCUGAU AUCAGAGUAAAUCUCACAGCCUG 397 398 1637-1659 UGCCAAAAAUGGACAUCAUUU AAAUGAUGUCCAUUUUUGGCAUG 399 400 1363-1385 AUGAUCCAAGGGAUUCGAAAC GUUUCGAAUCCCUUGGAUCAUGG 401 402 925-947 ACUUUUCAGUAUGAUCGUUUC GAAACGAUCAUACUGAAAAGUGG 403 404 790-812 CCCAGUGUGGUUAGUGUGAAA UUUCACACUAACCACACUGGGGC 405 406 1531-1553 UGUGAUGUGGCCCAUGAGUUU AAACUCAUGGGCCACAUCACACA 407 408 2189-2211 AUUUUGAAGUGAUGAGUGAAA UUUCACUCAUCACUUCAAAAUGC 409 410 929-951 UUCAGUAUGAUCGUUUCUUUG CAAAGAAACGAUCAUACUGAAAA 411 412 872-894 GACCAGAAAGAGUGUCUCAUC GAUGAGACACUCUUUCUGGUCUU 413 414 706-728 UUCUGCAAAGCCAGUCUUGAG CUCAAGACUGGCUUUGCAGAAGA 415 416 1362-1384 CAUGAUCCAAGGGAUUCGAAA UUUCGAAUCCCUUGGAUCAUGGA 417 418 1634-1656 UCAUGCCAAAAAUGGACAUCA UGAUGUCCAUUUUUGGCAUGACU 419 420 1325-1347 GCUGUGAGAUUUACUCUGAUU AAUCAGAGUAAAUCUCACAGCCU 421 422 2208-2230 AAGAGAGAAGUCCUAUUUCUC GAGAAAUAGGACUUCUCUCUUUC 423 424 2344-2366 AGUUAUAUUAAAUUUUAAUCU AGAUUAAAAUUUAAUAUAACUUA 425 426 924-946 CACUUUUCAGUAUGAUCGUUU AAACGAUCAUACUGAAAAGUGGA 427 428 873-895 ACCAGAAAGAGUGUCUCAUCU AGAUGAGACACUCUUUCUGGUCU 429 430 759-781 GAGGAAAGAGGUUGCUGAAAC GUUUCAGCAACCUCUUUCCUCAC 431 432 871-893 AGACCAGAAAGAGUGUCUCAU AUGAGACACUCUUUCUGGUCUUU 433 434 1183-1205 AAUAUUUCUGGAACUAGUAAA UUUACUAGUUCCAGAAAUAUUUC 435 436 2229-2251 AGGCUUGAGCAAGUUGGUAUC GAUACCAACUUGCUCAAGCCUGA 437 438 671-693 UGGCAGCACAGAUGAAUCAGA UCUGAUUCAUCUGUGCUGCCAGG 439 440 2187-2209 GCAUUUUGAAGUGAUGAGUGA UCACUCAUCACUUCAAAAUGCAG 441 442 913-935 AAAUCUGUUUCCACUUUUCAG CUGAAAAGUGGAAACAGAUUUUG 443 444 1977-1999 ACUAAUGAGCAGACAUAACAU AUGUUAUGUCUGCUCAUUAGUUC 445 446 1174-1196 GGUACUAGAAAUAUUUCUGGA UCCAGAAAUAUUUCUAGUACCAC 447 448 1810-1832 AUCCUGAAGAGCGCUGAGGGA UCCCUCAGCGCUCUUCAGGAUCC 449 450 892-914 CUUCUUCAAGAUAACUUGCCA UGGCAAGUUAUCUUGAAGAAGAU 451 452 877-899 GAAAGAGUGUCUCAUCUUCUU AAGAAGAUGAGACACUCUUUCUG 453 454 935-957 AUGAUCGUUUCUUUGAGAAAA UUUUCUCAAAGAAACGAUCAUAC 455 456 1975-1997 GAACUAAUGAGCAGACAUAAC GUUAUGUCUGCUCAUUAGUUCAU 457 458 1478-1500 CAUUUGAAACUGUCCAUUCAA UUGAAUGGACAGUUUCAAAUGCC 459 460 2366-2388 UAGUAAAAACAUAGUCCUGGA UCCAGGACUAUGUUUUUACUAUA 461 462 853-875 GACAUCAUGCAAAAGCAAAGA UCUUUGCUUUUGCAUGAUGUCCU 463 464 1966-1988 GUCUGUGAUGAACUAAUGAGC GCUCAUUAGUUCAUCACAGACUU 465 466 928-950 UUUCAGUAUGAUCGUUUCUUU AAAGAAACGAUCAUACUGAAAAG 467 468 1186-1208 AUUUCUGGAACUAGUAAAUUC GAAUUUACUAGUUCCAGAAAUAU 469 470 1189-1211 UCUGGAACUAGUAAAUUCCAU AUGGAAUUUACUAGUUCCAGAAA 471 472 973-995 AAUGACCACACCUAUCGAGUU AACUCGAUAGGUGUGGUCAUUCU 473 474 983-1005 CCUAUCGAGUUUUUAAAACUG CAGUUUUAAAAACUCGAUAGGUG 475 476 1185-1207 UAUUUCUGGAACUAGUAAAUU AAUUUACUAGUUCCAGAAAUAUU 477 478 2353-2375 AAAUUUUAAUCUAUAGUAAAA UUUUACUAUAGAUUAAAAUUUAA 479 480 875-897 CAGAAAGAGUGUCUCAUCUUC GAAGAUGAGACACUCUUUCUGGU 481 482 360-378 GCCCAUUCUUAUCCCGAGU ACUCGGGAUAAGAAUGGGC 483 484 428-446 CAAAACUGCCCCAAGAUGA UCAUCUUGGGGCAGUUUUG 485 486 873-891 CAGAAAGAGUGUCUCAUCU AGAUGAGACACUCUUUCUG 487 488 874-892 AGAAAGAGUGUCUCAUCUU AAGAUGAGACACUCUUUCU 489 490 877-895 AAGAGUGUCUCAUCUUCUU AAGAAGAUGAGACACUCUU 491 492 1295-1313 CUCUUCACCCUGGCUAAGA UCUUAGCCAGGGUGAAGAG 493 494 1296-1314 UCUUCACCCUGGCUAAGAU AUCUUAGCCAGGGUGAAGA 495 496 1299-1317 UCACCCUGGCUAAGAUGAU AUCAUCUUAGCCAGGGUGA 497 498 1347-1365 GGAACCAUGCCUCCAUGAU AUCAUGGAGGCAUGGUUCC 499 500 1355-1373 GCCUCCAUGAUCCAAGGGA UCCCUUGGAUCAUGGAGGC 501 502 1356-1374 CCUCCAUGAUCCAAGGGAU AUCCCUUGGAUCAUGGAGG 503 504 1357-1375 CUCCAUGAUCCAAGGGAUU AAUCCCUUGGAUCAUGGAG 505 506 1631-1649 GUCAUGCCAAAAAUGGACA UGUCCAUUUUUGGCAUGAC 507 508 1634-1652 AUGCCAAAAAUGGACAUCA UGAUGUCCAUUUUUGGCAU 509 510 1635-1653 UGCCAAAAAUGGACAUCAU AUGAUGUCCAUUUUUGGCA 511 512 1791-1809 CCCUGGAGUCUGUGCGGAU AUCCGCACAGACUCCAGGG 513 514 1794-1812 UGGAGUCUGUGCGGAUCCU AGGAUCCGCACAGACUCCA 515 516 1921-1939 CAUCAUCCCUGUGCGGGUU AACCCGCACAGGGAUGAUG 517 518 359-377 UGCCCAUUCUUAUCCCGAA UUCGGGAUAAGAAUGGGCA 519 520 362-380 CCAUUCUUAUCCCGAGUCA UGACUCGGGAUAAGAAUGG 521 522 363-381 CAUUCUUAUCCCGAGUCCA UGGACUCGGGAUAAGAAUG 523 524 434-452 UGCCCCAAGAUGAUGGAAU AUUCCAUCAUCUUGGGGCA 525 526 872-890 CCAGAAAGAGUGUCUCAUA UAUGAGACACUCUUUCUGG 527 528 875-893 GAAAGAGUGUCUCAUCUUA UAAGAUGAGACACUCUUUC 529 530 1112-1130 CACCCACGGGUGUGUGGGA UCCCACACACCCGUGGGUG 531 532 1113-1131 ACCCACGGGUGUGUGGGGA UCCCCACACACCCGUGGGU 533 534 1297-1315 CUUCACCCUGGCUAAGAUA UAUCUUAGCCAGGGUGAAG 535 536 1300-1318 CACCCUGGCUAAGAUGAUA UAUCAUCUUAGCCAGGGUG 537 538 1301-1319 ACCCUGGCUAAGAUGAUGA UCAUCAUCUUAGCCAGGGU 539 540 1348-1366 GAACCAUGCCUCCAUGAUA UAUCAUGGAGGCAUGGUUC 541 542 1481-1499 GAAACUGUCCAUUCAAUGA UCAUUGAAUGGACAGUUUC 543 544 1786-1804 UGGAGCCCUGGAGUCUGUA UACAGACUCCAGGGCUCCA 545 546 1795-1813 GGAGUCUGUGCGGAUCCUA UAGGAUCCGCACAGACUCC 547 548 1919-1937 CACAUCAUCCCUGUGCGGA UCCGCACAGGGAUGAUGUG 549 550 1922-1940 AUCAUCCCUGUGCGGGUUA UAACCCGCACAGGGAUGAU 551 552 1923-1941 UCAUCCCUGUGCGGGUUGA UCAACCCGCACAGGGAUGA

Example 8. Human siRNAs

Additional 19mer human siRNAs were generated. The sequences of these siRNAs are provided in Table 9. These siRNAs can be tested for efficacy using methods described herein and/or methods known in the art.

TABLE 9 Human ALAS1 siRNA Sense and Antisense Sequences SEQ SEQ ID ID NO: Position on NO: (anti- transcript Sense Sequence Antisense Sequence (sense) sense) NM_000688.4 (5′-3′) (5′-3′) 553 554  4-22 UAUAUUAAGGCGCCGGCGA UCGCCGGCGCCUUAAUAUA 555 556  5-23 AUAUUAAGGCGCCGGCGAU AUCGCCGGCGCCUUAAUAU 557 558  6-24 UAUUAAGGCGCCGGCGAUC GAUCGCCGGCGCCUUAAUA 559 560  7-25 AUUAAGGCGCCGGCGAUCG CGAUCGCCGGCGCCUUAAU 561 562  8-26 UUAAGGCGCCGGCGAUCGC GCGAUCGCCGGCGCCUUAA 563 564  9-27 UAAGGCGCCGGCGAUCGCG CGCGAUCGCCGGCGCCUUA 565 566 10-28 AAGGCGCCGGCGAUCGCGG CCGCGAUCGCCGGCGCCUU 567 568 11-29 AGGCGCCGGCGAUCGCGGC GCCGCGAUCGCCGGCGCCU 569 570 12-30 GGCGCCGGCGAUCGCGGCC GGCCGCGAUCGCCGGCGCC 571 572 13-31 GCGCCGGCGAUCGCGGCCU AGGCCGCGAUCGCCGGCGC 573 574 14-32 CGCCGGCGAUCGCGGCCUG CAGGCCGCGAUCGCCGGCG 575 576 81-99 CUUGAGUGCCCGCCUCCUU AAGGAGGCGGGCACUCAAG 577 578  82-100 UUGAGUGCCCGCCUCCUUC GAAGGAGGCGGGCACUCAA 579 580  83-101 UGAGUGCCCGCCUCCUUCG CGAAGGAGGCGGGCACUCA 581 582  84-102 GAGUGCCCGCCUCCUUCGC GCGAAGGAGGCGGGCACUC 583 584  85-103 AGUGCCCGCCUCCUUCGCC GGCGAAGGAGGCGGGCACU 585 586  86-104 GUGCCCGCCUCCUUCGCCG CGGCGAAGGAGGCGGGCAC 587 588  87-105 UGCCCGCCUCCUUCGCCGC GCGGCGAAGGAGGCGGGCA 589 590  88-106 GCCCGCCUCCUUCGCCGCC GGCGGCGAAGGAGGCGGGC 591 592  89-107 CCCGCCUCCUUCGCCGCCG CGGCGGCGAAGGAGGCGGG 593 594  90-108 CCGCCUCCUUCGCCGCCGC GCGGCGGCGAAGGAGGCGG 595 596  91-109 CGCCUCCUUCGCCGCCGCC GGCGGCGGCGAAGGAGGCG 597 598  92-110 GCCUCCUUCGCCGCCGCCU AGGCGGCGGCGAAGGAGGC 599 600  93-111 CCUCCUUCGCCGCCGCCUC GAGGCGGCGGCGAAGGAGG 601 602 356-374 CGCUGCCCAUUCUUAUCCC GGGAUAAGAAUGGGCAGCG 603 604 357-375 GCUGCCCAUUCUUAUCCCG CGGGAUAAGAAUGGGCAGC 605 606 359-377 UGCCCAUUCUUAUCCCGAG CUCGGGAUAAGAAUGGGCA 607 608 361-379 CCCAUUCUUAUCCCGAGUC GACUCGGGAUAAGAAUGGG 609 610 362-380 CCAUUCUUAUCCCGAGUCC GGACUCGGGAUAAGAAUGG 611 612 363-381 CAUUCUUAUCCCGAGUCCC GGGACUCGGGAUAAGAAUG 613 614 364-382 AUUCUUAUCCCGAGUCCCC GGGGACUCGGGAUAAGAAU 615 616 365-383 UUCUUAUCCCGAGUCCCCC GGGGGACUCGGGAUAAGAA 617 618 366-384 UCUUAUCCCGAGUCCCCCA UGGGGGACUCGGGAUAAGA 619 620 367-385 CUUAUCCCGAGUCCCCCAG CUGGGGGACUCGGGAUAAG 621 622 368-386 UUAUCCCGAGUCCCCCAGG CCUGGGGGACUCGGGAUAA 623 624 369-387 UAUCCCGAGUCCCCCAGGC GCCUGGGGGACUCGGGAUA 625 626 370-388 AUCCCGAGUCCCCCAGGCC GGCCUGGGGGACUCGGGAU 627 628 371-389 UCCCGAGUCCCCCAGGCCU AGGCCUGGGGGACUCGGGA 629 630 372-390 CCCGAGUCCCCCAGGCCUU AAGGCCUGGGGGACUCGGG 631 632 373-391 CCGAGUCCCCCAGGCCUUU AAAGGCCUGGGGGACUCGG 633 634 374-392 CGAGUCCCCCAGGCCUUUC GAAAGGCCUGGGGGACUCG 635 636 375-393 GAGUCCCCCAGGCCUUUCU AGAAAGGCCUGGGGGACUC 637 638 376-394 AGUCCCCCAGGCCUUUCUG CAGAAAGGCCUGGGGGACU 639 640 377-395 GUCCCCCAGGCCUUUCUGC GCAGAAAGGCCUGGGGGAC 641 642 378-396 UCCCCCAGGCCUUUCUGCA UGCAGAAAGGCCUGGGGGA 643 644 379-397 CCCCCAGGCCUUUCUGCAG CUGCAGAAAGGCCUGGGGG 645 646 380-398 CCCCAGGCCUUUCUGCAGA UCUGCAGAAAGGCCUGGGG 647 648 381-399 CCCAGGCCUUUCUGCAGAA UUCUGCAGAAAGGCCUGGG 649 650 382-400 CCAGGCCUUUCUGCAGAAA UUUCUGCAGAAAGGCCUGG 651 652 383-401 CAGGCCUUUCUGCAGAAAG CUUUCUGCAGAAAGGCCUG 653 654 384-402 AGGCCUUUCUGCAGAAAGC GCUUUCUGCAGAAAGGCCU 655 656 385-403 GGCCUUUCUGCAGAAAGCA UGCUUUCUGCAGAAAGGCC 657 658 386-404 GCCUUUCUGCAGAAAGCAG CUGCUUUCUGCAGAAAGGC 659 660 387-405 CCUUUCUGCAGAAAGCAGG CCUGCUUUCUGCAGAAAGG 661 662 388-406 CUUUCUGCAGAAAGCAGGC GCCUGCUUUCUGCAGAAAG 663 664 389-407 UUUCUGCAGAAAGCAGGCA UGCCUGCUUUCUGCAGAAA 665 666 390-408 UUCUGCAGAAAGCAGGCAA UUGCCUGCUUUCUGCAGAA 667 668 391-409 UCUGCAGAAAGCAGGCAAA UUUGCCUGCUUUCUGCAGA 669 670 392-410 CUGCAGAAAGCAGGCAAAU AUUUGCCUGCUUUCUGCAG 671 672 393-411 UGCAGAAAGCAGGCAAAUC GAUUUGCCUGCUUUCUGCA 673 674 394-412 GCAGAAAGCAGGCAAAUCU AGAUUUGCCUGCUUUCUGC 675 676 395-413 CAGAAAGCAGGCAAAUCUC GAGAUUUGCCUGCUUUCUG 677 678 396-414 AGAAAGCAGGCAAAUCUCU AGAGAUUUGCCUGCUUUCU 679 680 397-415 GAAAGCAGGCAAAUCUCUG CAGAGAUUUGCCUGCUUUC 681 682 398-416 AAAGCAGGCAAAUCUCUGU ACAGAGAUUUGCCUGCUUU 683 684 399-417 AAGCAGGCAAAUCUCUGUU AACAGAGAUUUGCCUGCUU 685 686 400-418 AGCAGGCAAAUCUCUGUUG CAACAGAGAUUUGCCUGCU 687 688 401-419 GCAGGCAAAUCUCUGUUGU ACAACAGAGAUUUGCCUGC 689 690 402-420 CAGGCAAAUCUCUGUUGUU AACAACAGAGAUUUGCCUG 691 692 403-421 AGGCAAAUCUCUGUUGUUC GAACAACAGAGAUUUGCCU 693 694 405-423 GCAAAUCUCUGUUGUUCUA UAGAACAACAGAGAUUUGC 695 696 406-424 CAAAUCUCUGUUGUUCUAU AUAGAACAACAGAGAUUUG 697 698 407-425 AAAUCUCUGUUGUUCUAUG CAUAGAACAACAGAGAUUU 699 700 408-426 AAUCUCUGUUGUUCUAUGC GCAUAGAACAACAGAGAUU 701 702 409-427 AUCUCUGUUGUUCUAUGCC GGCAUAGAACAACAGAGAU 703 704 410-428 UCUCUGUUGUUCUAUGCCC GGGCAUAGAACAACAGAGA 705 706 411-429 CUCUGUUGUUCUAUGCCCA UGGGCAUAGAACAACAGAG 707 708 412-430 UCUGUUGUUCUAUGCCCAA UUGGGCAUAGAACAACAGA 709 710 413-431 CUGUUGUUCUAUGCCCAAA UUUGGGCAUAGAACAACAG 711 712 414-432 UGUUGUUCUAUGCCCAAAA UUUUGGGCAUAGAACAACA 713 714 415-433 GUUGUUCUAUGCCCAAAAC GUUUUGGGCAUAGAACAAC 715 716 416-434 UUGUUCUAUGCCCAAAACU AGUUUUGGGCAUAGAACAA 717 718 417-435 UGUUCUAUGCCCAAAACUG CAGUUUUGGGCAUAGAACA 719 720 418-436 GUUCUAUGCCCAAAACUGC GCAGUUUUGGGCAUAGAAC 721 722 419-437 UUCUAUGCCCAAAACUGCC GGCAGUUUUGGGCAUAGAA 723 724 420-438 UCUAUGCCCAAAACUGCCC GGGCAGUUUUGGGCAUAGA 725 726 421-439 CUAUGCCCAAAACUGCCCC GGGGCAGUUUUGGGCAUAG 727 728 422-440 UAUGCCCAAAACUGCCCCA UGGGGCAGUUUUGGGCAUA 729 730 423-441 AUGCCCAAAACUGCCCCAA UUGGGGCAGUUUUGGGCAU 731 732 424-442 UGCCCAAAACUGCCCCAAG CUUGGGGCAGUUUUGGGCA 733 734 425-443 GCCCAAAACUGCCCCAAGA UCUUGGGGCAGUUUUGGGC 735 736 426-444 CCCAAAACUGCCCCAAGAU AUCUUGGGGCAGUUUUGGG 737 738 427-445 CCAAAACUGCCCCAAGAUG CAUCUUGGGGCAGUUUUGG 739 740 429-447 AAAACUGCCCCAAGAUGAU AUCAUCUUGGGGCAGUUUU 741 742 430-448 AAACUGCCCCAAGAUGAUG CAUCAUCUUGGGGCAGUUU 743 744 431-449 AACUGCCCCAAGAUGAUGG CCAUCAUCUUGGGGCAGUU 745 746 432-450 ACUGCCCCAAGAUGAUGGA UCCAUCAUCUUGGGGCAGU 747 748 433-451 CUGCCCCAAGAUGAUGGAA UUCCAUCAUCUUGGGGCAG 749 750 434-452 UGCCCCAAGAUGAUGGAAG CUUCCAUCAUCUUGGGGCA 751 752 435-453 GCCCCAAGAUGAUGGAAGU ACUUCCAUCAUCUUGGGGC 753 754 437-455 CCCAAGAUGAUGGAAGUUG CAACUUCCAUCAUCUUGGG 755 756 438-456 CCAAGAUGAUGGAAGUUGG CCAACUUCCAUCAUCUUGG 757 758 439-457 CAAGAUGAUGGAAGUUGGG CCCAACUUCCAUCAUCUUG 759 760 440-458 AAGAUGAUGGAAGUUGGGG CCCCAACUUCCAUCAUCUU 761 762 441-459 AGAUGAUGGAAGUUGGGGC GCCCCAACUUCCAUCAUCU 763 764 442-460 GAUGAUGGAAGUUGGGGCC GGCCCCAACUUCCAUCAUC 765 766 443-461 AUGAUGGAAGUUGGGGCCA UGGCCCCAACUUCCAUCAU 767 768 444-462 UGAUGGAAGUUGGGGCCAA UUGGCCCCAACUUCCAUCA 769 770 445-463 GAUGGAAGUUGGGGCCAAG CUUGGCCCCAACUUCCAUC 771 772 446-464 AUGGAAGUUGGGGCCAAGC GCUUGGCCCCAACUUCCAU 773 774 447-465 UGGAAGUUGGGGCCAAGCC GGCUUGGCCCCAACUUCCA 775 776 448-466 GGAAGUUGGGGCCAAGCCA UGGCUUGGCCCCAACUUCC 777 778 449-467 GAAGUUGGGGCCAAGCCAG CUGGCUUGGCCCCAACUUC 779 780 450-468 AAGUUGGGGCCAAGCCAGC GCUGGCUUGGCCCCAACUU 781 782 451-469 AGUUGGGGCCAAGCCAGCC GGCUGGCUUGGCCCCAACU 783 784 452-470 GUUGGGGCCAAGCCAGCCC GGGCUGGCUUGGCCCCAAC 785 786 453-471 UUGGGGCCAAGCCAGCCCC GGGGCUGGCUUGGCCCCAA 787 788 454-472 UGGGGCCAAGCCAGCCCCU AGGGGCUGGCUUGGCCCCA 789 790 455-473 GGGGCCAAGCCAGCCCCUC GAGGGGCUGGCUUGGCCCC 791 792 456-474 GGGCCAAGCCAGCCCCUCG CGAGGGGCUGGCUUGGCCC 793 794 457-475 GGCCAAGCCAGCCCCUCGG CCGAGGGGCUGGCUUGGCC 795 796 458-476 GCCAAGCCAGCCCCUCGGG CCCGAGGGGCUGGCUUGGC 797 798 459-477 CCAAGCCAGCCCCUCGGGC GCCCGAGGGGCUGGCUUGG 799 800 460-478 CAAGCCAGCCCCUCGGGCA UGCCCGAGGGGCUGGCUUG 801 802 461-479 AAGCCAGCCCCUCGGGCAU AUGCCCGAGGGGCUGGCUU 803 804 462-480 AGCCAGCCCCUCGGGCAUU AAUGCCCGAGGGGCUGGCU 805 806 463-481 GCCAGCCCCUCGGGCAUUG CAAUGCCCGAGGGGCUGGC 807 808 464-482 CCAGCCCCUCGGGCAUUGU ACAAUGCCCGAGGGGCUGG 809 810 465-483 CAGCCCCUCGGGCAUUGUC GACAAUGCCCGAGGGGCUG 811 812 466-484 AGCCCCUCGGGCAUUGUCC GGACAAUGCCCGAGGGGCU 813 814 467-485 GCCCCUCGGGCAUUGUCCA UGGACAAUGCCCGAGGGGC 815 816 468-486 CCCCUCGGGCAUUGUCCAC GUGGACAAUGCCCGAGGGG 817 818 469-487 CCCUCGGGCAUUGUCCACU AGUGGACAAUGCCCGAGGG 819 820 470-488 CCUCGGGCAUUGUCCACUG CAGUGGACAAUGCCCGAGG 821 822 471-489 CUCGGGCAUUGUCCACUGC GCAGUGGACAAUGCCCGAG 823 824 472-490 UCGGGCAUUGUCCACUGCA UGCAGUGGACAAUGCCCGA 825 826 473-491 CGGGCAUUGUCCACUGCAG CUGCAGUGGACAAUGCCCG 827 828 474-492 GGGCAUUGUCCACUGCAGC GCUGCAGUGGACAAUGCCC 829 830 475-493 GGCAUUGUCCACUGCAGCA UGCUGCAGUGGACAAUGCC 831 832 476-494 GCAUUGUCCACUGCAGCAG CUGCUGCAGUGGACAAUGC 833 834 477-495 CAUUGUCCACUGCAGCAGU ACUGCUGCAGUGGACAAUG 835 836 478-496 AUUGUCCACUGCAGCAGUA UACUGCUGCAGUGGACAAU 837 838 479-497 UUGUCCACUGCAGCAGUAC GUACUGCUGCAGUGGACAA 839 840 480-498 UGUCCACUGCAGCAGUACA UGUACUGCUGCAGUGGACA 841 842 481-499 GUCCACUGCAGCAGUACAC GUGUACUGCUGCAGUGGAC 843 844 482-500 UCCACUGCAGCAGUACACU AGUGUACUGCUGCAGUGGA 845 846 483-501 CCACUGCAGCAGUACACUA UAGUGUACUGCUGCAGUGG 847 848 484-502 CACUGCAGCAGUACACUAC GUAGUGUACUGCUGCAGUG 849 850 485-503 ACUGCAGCAGUACACUACC GGUAGUGUACUGCUGCAGU 851 852 486-504 CUGCAGCAGUACACUACCA UGGUAGUGUACUGCUGCAG 853 854 487-505 UGCAGCAGUACACUACCAA UUGGUAGUGUACUGCUGCA 855 856 488-506 GCAGCAGUACACUACCAAC GUUGGUAGUGUACUGCUGC 857 858 490-508 AGCAGUACACUACCAACAG CUGUUGGUAGUGUACUGCU 859 860 491-509 GCAGUACACUACCAACAGA UCUGUUGGUAGUGUACUGC 861 862 492-510 CAGUACACUACCAACAGAU AUCUGUUGGUAGUGUACUG 863 864 493-511 AGUACACUACCAACAGAUC GAUCUGUUGGUAGUGUACU 865 866 494-512 GUACACUACCAACAGAUCA UGAUCUGUUGGUAGUGUAC 867 868 495-513 UACACUACCAACAGAUCAA UUGAUCUGUUGGUAGUGUA 869 870 496-514 ACACUACCAACAGAUCAAA UUUGAUCUGUUGGUAGUGU 871 872 497-515 CACUACCAACAGAUCAAAG CUUUGAUCUGUUGGUAGUG 873 874 498-516 ACUACCAACAGAUCAAAGA UCUUUGAUCUGUUGGUAGU 875 876 499-517 CUACCAACAGAUCAAAGAA UUCUUUGAUCUGUUGGUAG 877 878 500-518 UACCAACAGAUCAAAGAAA UUUCUUUGAUCUGUUGGUA 879 880 501-519 ACCAACAGAUCAAAGAAAC GUUUCUUUGAUCUGUUGGU 881 882 502-520 CCAACAGAUCAAAGAAACC GGUUUCUUUGAUCUGUUGG 883 884 523-541 UCCGGCCAGUGAGAAAGAC GUCUUUCUCACUGGCCGGA 885 886 524-542 CCGGCCAGUGAGAAAGACA UGUCUUUCUCACUGGCCGG 887 888 525-543 CGGCCAGUGAGAAAGACAA UUGUCUUUCUCACUGGCCG 889 890 526-544 GGCCAGUGAGAAAGACAAA UUUGUCUUUCUCACUGGCC 891 892 527-545 GCCAGUGAGAAAGACAAAA UUUUGUCUUUCUCACUGGC 893 894 528-546 CCAGUGAGAAAGACAAAAC GUUUUGUCUUUCUCACUGG 895 896 529-547 CAGUGAGAAAGACAAAACU AGUUUUGUCUUUCUCACUG 897 898 530-548 AGUGAGAAAGACAAAACUG CAGUUUUGUCUUUCUCACU 899 900 531-549 GUGAGAAAGACAAAACUGC GCAGUUUUGUCUUUCUCAC 901 902 570-588 CUCCUGAUGGAUCCCAGCA UGCUGGGAUCCAUCAGGAG 903 904 571-589 UCCUGAUGGAUCCCAGCAG CUGCUGGGAUCCAUCAGGA 905 906 572-590 CCUGAUGGAUCCCAGCAGA UCUGCUGGGAUCCAUCAGG 907 908 573-591 CUGAUGGAUCCCAGCAGAG CUCUGCUGGGAUCCAUCAG 909 910 574-592 UGAUGGAUCCCAGCAGAGU ACUCUGCUGGGAUCCAUCA 911 912 575-593 GAUGGAUCCCAGCAGAGUC GACUCUGCUGGGAUCCAUC 913 914 576-594 AUGGAUCCCAGCAGAGUCC GGACUCUGCUGGGAUCCAU 915 916 577-595 UGGAUCCCAGCAGAGUCCA UGGACUCUGCUGGGAUCCA 917 918 578-596 GGAUCCCAGCAGAGUCCAG CUGGACUCUGCUGGGAUCC 919 920 579-597 GAUCCCAGCAGAGUCCAGA UCUGGACUCUGCUGGGAUC 921 922 580-598 AUCCCAGCAGAGUCCAGAU AUCUGGACUCUGCUGGGAU 923 924 581-599 UCCCAGCAGAGUCCAGAUG CAUCUGGACUCUGCUGGGA 925 926 582-600 CCCAGCAGAGUCCAGAUGG CCAUCUGGACUCUGCUGGG 927 928 583-601 CCAGCAGAGUCCAGAUGGC GCCAUCUGGACUCUGCUGG 929 930 584-602 CAGCAGAGUCCAGAUGGCA UGCCAUCUGGACUCUGCUG 931 932 585-603 AGCAGAGUCCAGAUGGCAC GUGCCAUCUGGACUCUGCU 933 934 586-604 GCAGAGUCCAGAUGGCACA UGUGCCAUCUGGACUCUGC 935 936 587-605 CAGAGUCCAGAUGGCACAC GUGUGCCAUCUGGACUCUG 937 938 588-606 AGAGUCCAGAUGGCACACA UGUGUGCCAUCUGGACUCU 939 940 589-607 GAGUCCAGAUGGCACACAG CUGUGUGCCAUCUGGACUC 941 942 590-608 AGUCCAGAUGGCACACAGC GCUGUGUGCCAUCUGGACU 943 944 591-609 GUCCAGAUGGCACACAGCU AGCUGUGUGCCAUCUGGAC 945 946 592-610 UCCAGAUGGCACACAGCUU AAGCUGUGUGCCAUCUGGA 947 948 593-611 CCAGAUGGCACACAGCUUC GAAGCUGUGUGCCAUCUGG 949 950 594-612 CAGAUGGCACACAGCUUCC GGAAGCUGUGUGCCAUCUG 951 952 595-613 AGAUGGCACACAGCUUCCG CGGAAGCUGUGUGCCAUCU 953 954 596-614 GAUGGCACACAGCUUCCGU ACGGAAGCUGUGUGCCAUC 955 956 597-615 AUGGCACACAGCUUCCGUC GACGGAAGCUGUGUGCCAU 957 958 598-616 UGGCACACAGCUUCCGUCU AGACGGAAGCUGUGUGCCA 959 960 599-617 GGCACACAGCUUCCGUCUG CAGACGGAAGCUGUGUGCC 961 962 600-618 GCACACAGCUUCCGUCUGG CCAGACGGAAGCUGUGUGC 963 964 601-619 CACACAGCUUCCGUCUGGA UCCAGACGGAAGCUGUGUG 965 966 602-620 ACACAGCUUCCGUCUGGAC GUCCAGACGGAAGCUGUGU 967 968 603-621 CACAGCUUCCGUCUGGACA UGUCCAGACGGAAGCUGUG 969 970 604-622 ACAGCUUCCGUCUGGACAC GUGUCCAGACGGAAGCUGU 971 972 605-623 CAGCUUCCGUCUGGACACC GGUGUCCAGACGGAAGCUG 973 974 606-624 AGCUUCCGUCUGGACACCC GGGUGUCCAGACGGAAGCU 975 976 607-625 GCUUCCGUCUGGACACCCC GGGGUGUCCAGACGGAAGC 977 978 608-626 CUUCCGUCUGGACACCCCU AGGGGUGUCCAGACGGAAG 979 980 609-627 UUCCGUCUGGACACCCCUU AAGGGGUGUCCAGACGGAA 981 982 610-628 UCCGUCUGGACACCCCUUG CAAGGGGUGUCCAGACGGA 983 984 611-629 CCGUCUGGACACCCCUUGC GCAAGGGGUGUCCAGACGG 985 986 612-630 CGUCUGGACACCCCUUGCC GGCAAGGGGUGUCCAGACG 987 988 613-631 GUCUGGACACCCCUUGCCU AGGCAAGGGGUGUCCAGAC 989 990 614-632 UCUGGACACCCCUUGCCUG CAGGCAAGGGGUGUCCAGA 991 992 615-633 CUGGACACCCCUUGCCUGC GCAGGCAAGGGGUGUCCAG 993 994 616-634 UGGACACCCCUUGCCUGCC GGCAGGCAAGGGGUGUCCA 995 996 617-635 GGACACCCCUUGCCUGCCA UGGCAGGCAAGGGGUGUCC 997 998 618-636 GACACCCCUUGCCUGCCAC GUGGCAGGCAAGGGGUGUC 999 1000 619-637 ACACCCCUUGCCUGCCACA UGUGGCAGGCAAGGGGUGU 1001 1002 620-638 CACCCCUUGCCUGCCACAA UUGUGGCAGGCAAGGGGUG 1003 1004 621-639 ACCCCUUGCCUGCCACAAG CUUGUGGCAGGCAAGGGGU 1005 1006 622-640 CCCCUUGCCUGCCACAAGC GCUUGUGGCAGGCAAGGGG 1007 1008 623-641 CCCUUGCCUGCCACAAGCC GGCUUGUGGCAGGCAAGGG 1009 1010 624-642 CCUUGCCUGCCACAAGCCA UGGCUUGUGGCAGGCAAGG 1011 1012 625-643 CUUGCCUGCCACAAGCCAG CUGGCUUGUGGCAGGCAAG 1013 1014 626-644 UUGCCUGCCACAAGCCAGG CCUGGCUUGUGGCAGGCAA 1015 1016 627-645 UGCCUGCCACAAGCCAGGG CCCUGGCUUGUGGCAGGCA 1017 1018 628-646 GCCUGCCACAAGCCAGGGC GCCCUGGCUUGUGGCAGGC 1019 1020 629-647 CCUGCCACAAGCCAGGGCA UGCCCUGGCUUGUGGCAGG 1021 1022 630-648 CUGCCACAAGCCAGGGCAC GUGCCCUGGCUUGUGGCAG 1023 1024 631-649 UGCCACAAGCCAGGGCACU AGUGCCCUGGCUUGUGGCA 1025 1026 632-650 GCCACAAGCCAGGGCACUG CAGUGCCCUGGCUUGUGGC 1027 1028 633-651 CCACAAGCCAGGGCACUGC GCAGUGCCCUGGCUUGUGG 1029 1030 634-652 CACAAGCCAGGGCACUGCA UGCAGUGCCCUGGCUUGUG 1031 1032 635-653 ACAAGCCAGGGCACUGCAA UUGCAGUGCCCUGGCUUGU 1033 1034 636-654 CAAGCCAGGGCACUGCAAG CUUGCAGUGCCCUGGCUUG 1035 1036 637-655 AAGCCAGGGCACUGCAAGC GCUUGCAGUGCCCUGGCUU 1037 1038 638-656 AGCCAGGGCACUGCAAGCA UGCUUGCAGUGCCCUGGCU 1039 1040 639-657 GCCAGGGCACUGCAAGCAA UUGCUUGCAGUGCCCUGGC 1041 1042 640-658 CCAGGGCACUGCAAGCAAA UUUGCUUGCAGUGCCCUGG 1043 1044 641-659 CAGGGCACUGCAAGCAAAU AUUUGCUUGCAGUGCCCUG 1045 1046 642-660 AGGGCACUGCAAGCAAAUG CAUUUGCUUGCAGUGCCCU 1047 1048 643-661 GGGCACUGCAAGCAAAUGC GCAUUUGCUUGCAGUGCCC 1049 1050 644-662 GGCACUGCAAGCAAAUGCC GGCAUUUGCUUGCAGUGCC 1051 1052 645-663 GCACUGCAAGCAAAUGCCC GGGCAUUUGCUUGCAGUGC 1053 1054 647-665 ACUGCAAGCAAAUGCCCUU AAGGGCAUUUGCUUGCAGU 1055 1056 648-666 CUGCAAGCAAAUGCCCUUU AAAGGGCAUUUGCUUGCAG 1057 1058 649-667 UGCAAGCAAAUGCCCUUUC GAAAGGGCAUUUGCUUGCA 1059 1060 650-668 GCAAGCAAAUGCCCUUUCC GGAAAGGGCAUUUGCUUGC 1061 1062 651-669 CAAGCAAAUGCCCUUUCCU AGGAAAGGGCAUUUGCUUG 1063 1064 652-670 AAGCAAAUGCCCUUUCCUG CAGGAAAGGGCAUUUGCUU 1065 1066 653-671 AGCAAAUGCCCUUUCCUGG CCAGGAAAGGGCAUUUGCU 1067 1068 654-672 GCAAAUGCCCUUUCCUGGC GCCAGGAAAGGGCAUUUGC 1069 1070 655-673 CAAAUGCCCUUUCCUGGCA UGCCAGGAAAGGGCAUUUG 1071 1072 656-674 AAAUGCCCUUUCCUGGCAG CUGCCAGGAAAGGGCAUUU 1073 1074 657-675 AAUGCCCUUUCCUGGCAGC GCUGCCAGGAAAGGGCAUU 1075 1076 658-676 AUGCCCUUUCCUGGCAGCA UGCUGCCAGGAAAGGGCAU 1077 1078 659-677 UGCCCUUUCCUGGCAGCAC GUGCUGCCAGGAAAGGGCA 1079 1080 660-678 GCCCUUUCCUGGCAGCACA UGUGCUGCCAGGAAAGGGC 1081 1082 661-679 CCCUUUCCUGGCAGCACAG CUGUGCUGCCAGGAAAGGG 1083 1084 662-680 CCUUUCCUGGCAGCACAGA UCUGUGCUGCCAGGAAAGG 1085 1086 663-681 CUUUCCUGGCAGCACAGAU AUCUGUGCUGCCAGGAAAG 1087 1088 664-682 UUUCCUGGCAGCACAGAUG CAUCUGUGCUGCCAGGAAA 1089 1090 665-683 UUCCUGGCAGCACAGAUGA UCAUCUGUGCUGCCAGGAA 1091 1092 666-684 UCCUGGCAGCACAGAUGAA UUCAUCUGUGCUGCCAGGA 1093 1094 667-685 CCUGGCAGCACAGAUGAAU AUUCAUCUGUGCUGCCAGG 1095 1096 668-686 CUGGCAGCACAGAUGAAUC GAUUCAUCUGUGCUGCCAG 1097 1098 670-688 GGCAGCACAGAUGAAUCAG CUGAUUCAUCUGUGCUGCC 1099 1100 672-690 CAGCACAGAUGAAUCAGAG CUCUGAUUCAUCUGUGCUG 1101 1102 692-710 GGCAGCAGUGUCUUCUGCA UGCAGAAGACACUGCUGCC 1103 1104 693-711 GCAGCAGUGUCUUCUGCAA UUGCAGAAGACACUGCUGC 1105 1106 694-712 CAGCAGUGUCUUCUGCAAA UUUGCAGAAGACACUGCUG 1107 1108 695-713 AGCAGUGUCUUCUGCAAAG CUUUGCAGAAGACACUGCU 1109 1110 696-714 GCAGUGUCUUCUGCAAAGC GCUUUGCAGAAGACACUGC 1111 1112 697-715 CAGUGUCUUCUGCAAAGCC GGCUUUGCAGAAGACACUG 1113 1114 698-716 AGUGUCUUCUGCAAAGCCA UGGCUUUGCAGAAGACACU 1115 1116 699-717 GUGUCUUCUGCAAAGCCAG CUGGCUUUGCAGAAGACAC 1117 1118 700-718 UGUCUUCUGCAAAGCCAGU ACUGGCUUUGCAGAAGACA 1119 1120 701-719 GUCUUCUGCAAAGCCAGUC GACUGGCUUUGCAGAAGAC 1121 1122 702-720 UCUUCUGCAAAGCCAGUCU AGACUGGCUUUGCAGAAGA 1123 1124 703-721 CUUCUGCAAAGCCAGUCUU AAGACUGGCUUUGCAGAAG 1125 1126 704-722 UUCUGCAAAGCCAGUCUUG CAAGACUGGCUUUGCAGAA 1127 1128 705-723 UCUGCAAAGCCAGUCUUGA UCAAGACUGGCUUUGCAGA 1129 1130 706-724 CUGCAAAGCCAGUCUUGAG CUCAAGACUGGCUUUGCAG 1131 1132 707-725 UGCAAAGCCAGUCUUGAGC GCUCAAGACUGGCUUUGCA 1133 1134 708-726 GCAAAGCCAGUCUUGAGCU AGCUCAAGACUGGCUUUGC 1135 1136 709-727 CAAAGCCAGUCUUGAGCUU AAGCUCAAGACUGGCUUUG 1137 1138 710-728 AAAGCCAGUCUUGAGCUUC GAAGCUCAAGACUGGCUUU 1139 1140 711-729 AAGCCAGUCUUGAGCUUCA UGAAGCUCAAGACUGGCUU 1141 1142 712-730 AGCCAGUCUUGAGCUUCAG CUGAAGCUCAAGACUGGCU 1143 1144 713-731 GCCAGUCUUGAGCUUCAGG CCUGAAGCUCAAGACUGGC 1145 1146 714-732 CCAGUCUUGAGCUUCAGGA UCCUGAAGCUCAAGACUGG 1147 1148 715-733 CAGUCUUGAGCUUCAGGAG CUCCUGAAGCUCAAGACUG 1149 1150 716-734 AGUCUUGAGCUUCAGGAGG CCUCCUGAAGCUCAAGACU 1151 1152 717-735 GUCUUGAGCUUCAGGAGGA UCCUCCUGAAGCUCAAGAC 1153 1154 718-736 UCUUGAGCUUCAGGAGGAU AUCCUCCUGAAGCUCAAGA 1155 1156 719-737 CUUGAGCUUCAGGAGGAUG CAUCCUCCUGAAGCUCAAG 1157 1158 720-738 UUGAGCUUCAGGAGGAUGU ACAUCCUCCUGAAGCUCAA 1159 1160 721-739 UGAGCUUCAGGAGGAUGUG CACAUCCUCCUGAAGCUCA 1161 1162 722-740 GAGCUUCAGGAGGAUGUGC GCACAUCCUCCUGAAGCUC 1163 1164 723-741 AGCUUCAGGAGGAUGUGCA UGCACAUCCUCCUGAAGCU 1165 1166 724-742 GCUUCAGGAGGAUGUGCAG CUGCACAUCCUCCUGAAGC 1167 1168 725-743 CUUCAGGAGGAUGUGCAGG CCUGCACAUCCUCCUGAAG 1169 1170 726-744 UUCAGGAGGAUGUGCAGGA UCCUGCACAUCCUCCUGAA 1171 1172 727-745 UCAGGAGGAUGUGCAGGAA UUCCUGCACAUCCUCCUGA 1173 1174 728-746 CAGGAGGAUGUGCAGGAAA UUUCCUGCACAUCCUCCUG 1175 1176 729-747 AGGAGGAUGUGCAGGAAAU AUUUCCUGCACAUCCUCCU 1177 1178 730-748 GGAGGAUGUGCAGGAAAUG CAUUUCCUGCACAUCCUCC 1179 1180 731-749 GAGGAUGUGCAGGAAAUGA UCAUUUCCUGCACAUCCUC 1181 1182 732-750 AGGAUGUGCAGGAAAUGAA UUCAUUUCCUGCACAUCCU 1183 1184 733-751 GGAUGUGCAGGAAAUGAAU AUUCAUUUCCUGCACAUCC 1185 1186 734-752 GAUGUGCAGGAAAUGAAUG CAUUCAUUUCCUGCACAUC 1187 1188 735-753 AUGUGCAGGAAAUGAAUGC GCAUUCAUUUCCUGCACAU 1189 1190 755-773 GUGAGGAAAGAGGUUGCUG CAGCAACCUCUUUCCUCAC 1191 1192 756-774 UGAGGAAAGAGGUUGCUGA UCAGCAACCUCUUUCCUCA 1193 1194 757-775 GAGGAAAGAGGUUGCUGAA UUCAGCAACCUCUUUCCUC 1195 1196 758-776 AGGAAAGAGGUUGCUGAAA UUUCAGCAACCUCUUUCCU 1197 1198 759-777 GGAAAGAGGUUGCUGAAAC GUUUCAGCAACCUCUUUCC 1199 1200 760-778 GAAAGAGGUUGCUGAAACC GGUUUCAGCAACCUCUUUC 1201 1202 761-779 AAAGAGGUUGCUGAAACCU AGGUUUCAGCAACCUCUUU 1203 1204 762-780 AAGAGGUUGCUGAAACCUC GAGGUUUCAGCAACCUCUU 1205 1206 763-781 AGAGGUUGCUGAAACCUCA UGAGGUUUCAGCAACCUCU 1207 1208 764-782 GAGGUUGCUGAAACCUCAG CUGAGGUUUCAGCAACCUC 1209 1210 765-783 AGGUUGCUGAAACCUCAGC GCUGAGGUUUCAGCAACCU 1211 1212 766-784 GGUUGCUGAAACCUCAGCA UGCUGAGGUUUCAGCAACC 1213 1214 787-805 CCCCAGUGUGGUUAGUGUG CACACUAACCACACUGGGG 1215 1216 791-809 AGUGUGGUUAGUGUGAAAA UUUUCACACUAACCACACU 1217 1218 792-810 GUGUGGUUAGUGUGAAAAC GUUUUCACACUAACCACAC 1219 1220 812-830 GAUGGAGGGGAUCCCAGUG CACUGGGAUCCCCUCCAUC 1221 1222 813-831 AUGGAGGGGAUCCCAGUGG CCACUGGGAUCCCCUCCAU 1223 1224 833-851 CUGCUGAAGAACUUCCAGG CCUGGAAGUUCUUCAGCAG 1225 1226 834-852 UGCUGAAGAACUUCCAGGA UCCUGGAAGUUCUUCAGCA 1227 1228 835-853 GCUGAAGAACUUCCAGGAC GUCCUGGAAGUUCUUCAGC 1229 1230 836-854 CUGAAGAACUUCCAGGACA UGUCCUGGAAGUUCUUCAG 1231 1232 837-855 UGAAGAACUUCCAGGACAU AUGUCCUGGAAGUUCUUCA 1233 1234 838-856 GAAGAACUUCCAGGACAUC GAUGUCCUGGAAGUUCUUC 1235 1236 839-857 AAGAACUUCCAGGACAUCA UGAUGUCCUGGAAGUUCUU 1237 1238 840-858 AGAACUUCCAGGACAUCAU AUGAUGUCCUGGAAGUUCU 1239 1240 841-859 GAACUUCCAGGACAUCAUG CAUGAUGUCCUGGAAGUUC 1241 1242 842-860 AACUUCCAGGACAUCAUGC GCAUGAUGUCCUGGAAGUU 1243 1244 843-861 ACUUCCAGGACAUCAUGCA UGCAUGAUGUCCUGGAAGU 1245 1246 844-862 CUUCCAGGACAUCAUGCAA UUGCAUGAUGUCCUGGAAG 1247 1248 845-863 UUCCAGGACAUCAUGCAAA UUUGCAUGAUGUCCUGGAA 1249 1250 846-864 UCCAGGACAUCAUGCAAAA UUUUGCAUGAUGUCCUGGA 1251 1252 847-865 CCAGGACAUCAUGCAAAAG CUUUUGCAUGAUGUCCUGG 1253 1254 848-866 CAGGACAUCAUGCAAAAGC GCUUUUGCAUGAUGUCCUG 1255 1256 849-867 AGGACAUCAUGCAAAAGCA UGCUUUUGCAUGAUGUCCU 1257 1258 850-868 GGACAUCAUGCAAAAGCAA UUGCUUUUGCAUGAUGUCC 1259 1260 851-869 GACAUCAUGCAAAAGCAAA UUUGCUUUUGCAUGAUGUC 1261 1262 852-870 ACAUCAUGCAAAAGCAAAG CUUUGCUUUUGCAUGAUGU 1263 1264 854-872 AUCAUGCAAAAGCAAAGAC GUCUUUGCUUUUGCAUGAU 1265 1266 855-873 UCAUGCAAAAGCAAAGACC GGUCUUUGCUUUUGCAUGA 1267 1268 856-874 CAUGCAAAAGCAAAGACCA UGGUCUUUGCUUUUGCAUG 1269 1270 857-875 AUGCAAAAGCAAAGACCAG CUGGUCUUUGCUUUUGCAU 1271 1272 858-876 UGCAAAAGCAAAGACCAGA UCUGGUCUUUGCUUUUGCA 1273 1274 859-877 GCAAAAGCAAAGACCAGAA UUCUGGUCUUUGCUUUUGC 1275 1276 860-878 CAAAAGCAAAGACCAGAAA UUUCUGGUCUUUGCUUUUG 1277 1278 861-879 AAAAGCAAAGACCAGAAAG CUUUCUGGUCUUUGCUUUU 1279 1280 862-880 AAAGCAAAGACCAGAAAGA UCUUUCUGGUCUUUGCUUU 1281 1282 863-881 AAGCAAAGACCAGAAAGAG CUCUUUCUGGUCUUUGCUU 1283 1284 864-882 AGCAAAGACCAGAAAGAGU ACUCUUUCUGGUCUUUGCU 1285 1286 865-883 GCAAAGACCAGAAAGAGUG CACUCUUUCUGGUCUUUGC 1287 1288 867-885 AAAGACCAGAAAGAGUGUC GACACUCUUUCUGGUCUUU 1289 1290 868-886 AAGACCAGAAAGAGUGUCU AGACACUCUUUCUGGUCUU 1291 1292 869-887 AGACCAGAAAGAGUGUCUC GAGACACUCUUUCUGGUCU 1293 1294 870-888 GACCAGAAAGAGUGUCUCA UGAGACACUCUUUCUGGUC 1295 1296 871-889 ACCAGAAAGAGUGUCUCAU AUGAGACACUCUUUCUGGU 1297 1298 872-890 CCAGAAAGAGUGUCUCAUC GAUGAGACACUCUUUCUGG 1299 1300 875-893 GAAAGAGUGUCUCAUCUUC GAAGAUGAGACACUCUUUC 1301 1302 878-896 AGAGUGUCUCAUCUUCUUC GAAGAAGAUGAGACACUCU 1303 1304 879-897 GAGUGUCUCAUCUUCUUCA UGAAGAAGAUGAGACACUC 1305 1306 880-898 AGUGUCUCAUCUUCUUCAA UUGAAGAAGAUGAGACACU 1307 1308 881-899 GUGUCUCAUCUUCUUCAAG CUUGAAGAAGAUGAGACAC 1309 1310 882-900 UGUCUCAUCUUCUUCAAGA UCUUGAAGAAGAUGAGACA 1311 1312 883-901 GUCUCAUCUUCUUCAAGAU AUCUUGAAGAAGAUGAGAC 1313 1314 884-902 UCUCAUCUUCUUCAAGAUA UAUCUUGAAGAAGAUGAGA 1315 1316 886-904 UCAUCUUCUUCAAGAUAAC GUUAUCUUGAAGAAGAUGA 1317 1318 887-905 CAUCUUCUUCAAGAUAACU AGUUAUCUUGAAGAAGAUG 1319 1320 888-906 AUCUUCUUCAAGAUAACUU AAGUUAUCUUGAAGAAGAU 1321 1322 889-907 UCUUCUUCAAGAUAACUUG CAAGUUAUCUUGAAGAAGA 1323 1324 890-908 CUUCUUCAAGAUAACUUGC GCAAGUUAUCUUGAAGAAG 1325 1326 891-909 UUCUUCAAGAUAACUUGCC GGCAAGUUAUCUUGAAGAA 1327 1328 892-910 UCUUCAAGAUAACUUGCCA UGGCAAGUUAUCUUGAAGA 1329 1330 893-911 CUUCAAGAUAACUUGCCAA UUGGCAAGUUAUCUUGAAG 1331 1332 894-912 UUCAAGAUAACUUGCCAAA UUUGGCAAGUUAUCUUGAA 1333 1334 895-913 UCAAGAUAACUUGCCAAAA UUUUGGCAAGUUAUCUUGA 1335 1336 896-914 CAAGAUAACUUGCCAAAAU AUUUUGGCAAGUUAUCUUG 1337 1338 897-915 AAGAUAACUUGCCAAAAUC GAUUUUGGCAAGUUAUCUU 1339 1340 898-916 AGAUAACUUGCCAAAAUCU AGAUUUUGGCAAGUUAUCU 1341 1342 899-917 GAUAACUUGCCAAAAUCUG CAGAUUUUGGCAAGUUAUC 1343 1344 900-918 AUAACUUGCCAAAAUCUGU ACAGAUUUUGGCAAGUUAU 1345 1346 901-919 UAACUUGCCAAAAUCUGUU AACAGAUUUUGGCAAGUUA 1347 1348 902-920 AACUUGCCAAAAUCUGUUU AAACAGAUUUUGGCAAGUU 1349 1350 903-921 ACUUGCCAAAAUCUGUUUC GAAACAGAUUUUGGCAAGU 1351 1352 904-922 CUUGCCAAAAUCUGUUUCC GGAAACAGAUUUUGGCAAG 1353 1354 905-923 UUGCCAAAAUCUGUUUCCA UGGAAACAGAUUUUGGCAA 1355 1356 906-924 UGCCAAAAUCUGUUUCCAC GUGGAAACAGAUUUUGGCA 1357 1358 907-925 GCCAAAAUCUGUUUCCACU AGUGGAAACAGAUUUUGGC 1359 1360 908-926 CCAAAAUCUGUUUCCACUU AAGUGGAAACAGAUUUUGG 1361 1362 909-927 CAAAAUCUGUUUCCACUUU AAAGUGGAAACAGAUUUUG 1363 1364 910-928 AAAAUCUGUUUCCACUUUU AAAAGUGGAAACAGAUUUU 1365 1366 911-929 AAAUCUGUUUCCACUUUUC GAAAAGUGGAAACAGAUUU 1367 1368 912-930 AAUCUGUUUCCACUUUUCA UGAAAAGUGGAAACAGAUU 1369 1370 913-931 AUCUGUUUCCACUUUUCAG CUGAAAAGUGGAAACAGAU 1371 1372 916-934 UGUUUCCACUUUUCAGUAU AUACUGAAAAGUGGAAACA 1373 1374 917-935 GUUUCCACUUUUCAGUAUG CAUACUGAAAAGUGGAAAC 1375 1376 918-936 UUUCCACUUUUCAGUAUGA UCAUACUGAAAAGUGGAAA 1377 1378 919-937 UUCCACUUUUCAGUAUGAU AUCAUACUGAAAAGUGGAA 1379 1380 920-938 UCCACUUUUCAGUAUGAUC GAUCAUACUGAAAAGUGGA 1381 1382 921-939 CCACUUUUCAGUAUGAUCG CGAUCAUACUGAAAAGUGG 1383 1384 925-943 UUUUCAGUAUGAUCGUUUC GAAACGAUCAUACUGAAAA 1385 1386 929-947 CAGUAUGAUCGUUUCUUUG CAAAGAAACGAUCAUACUG 1387 1388 930-948 AGUAUGAUCGUUUCUUUGA UCAAAGAAACGAUCAUACU 1389 1390 931-949 GUAUGAUCGUUUCUUUGAG CUCAAAGAAACGAUCAUAC 1391 1392 933-951 AUGAUCGUUUCUUUGAGAA UUCUCAAAGAAACGAUCAU 1393 1394 934-952 UGAUCGUUUCUUUGAGAAA UUUCUCAAAGAAACGAUCA 1395 1396 936-954 AUCGUUUCUUUGAGAAAAA UUUUUCUCAAAGAAACGAU 1397 1398 937-955 UCGUUUCUUUGAGAAAAAA UUUUUUCUCAAAGAAACGA 1399 1400 938-956 CGUUUCUUUGAGAAAAAAA UUUUUUUCUCAAAGAAACG 1401 1402 939-957 GUUUCUUUGAGAAAAAAAU AUUUUUUUCUCAAAGAAAC 1403 1404 940-958 UUUCUUUGAGAAAAAAAUU AAUUUUUUUCUCAAAGAAA 1405 1406 941-959 UUCUUUGAGAAAAAAAUUG CAAUUUUUUUCUCAAAGAA 1407 1408 942-960 UCUUUGAGAAAAAAAUUGA UCAAUUUUUUUCUCAAAGA 1409 1410 943-961 CUUUGAGAAAAAAAUUGAU AUCAAUUUUUUUCUCAAAG 1411 1412 944-962 UUUGAGAAAAAAAUUGAUG CAUCAAUUUUUUUCUCAAA 1413 1414 945-963 UUGAGAAAAAAAUUGAUGA UCAUCAAUUUUUUUCUCAA 1415 1416 946-964 UGAGAAAAAAAUUGAUGAG CUCAUCAAUUUUUUUCUCA 1417 1418 947-965 GAGAAAAAAAUUGAUGAGA UCUCAUCAAUUUUUUUCUC 1419 1420 948-966 AGAAAAAAAUUGAUGAGAA UUCUCAUCAAUUUUUUUCU 1421 1422 949-967 GAAAAAAAUUGAUGAGAAA UUUCUCAUCAAUUUUUUUC 1423 1424 950-968 AAAAAAAUUGAUGAGAAAA UUUUCUCAUCAAUUUUUUU 1425 1426 951-969 AAAAAAUUGAUGAGAAAAA UUUUUCUCAUCAAUUUUUU 1427 1428 952-970 AAAAAUUGAUGAGAAAAAG CUUUUUCUCAUCAAUUUUU 1429 1430 953-971 AAAAUUGAUGAGAAAAAGA UCUUUUUCUCAUCAAUUUU 1431 1432 954-972 AAAUUGAUGAGAAAAAGAA UUCUUUUUCUCAUCAAUUU 1433 1434 955-973 AAUUGAUGAGAAAAAGAAU AUUCUUUUUCUCAUCAAUU 1435 1436 956-974 AUUGAUGAGAAAAAGAAUG CAUUCUUUUUCUCAUCAAU 1437 1438 957-975 UUGAUGAGAAAAAGAAUGA UCAUUCUUUUUCUCAUCAA 1439 1440 958-976 UGAUGAGAAAAAGAAUGAC GUCAUUCUUUUUCUCAUCA 1441 1442 959-977 GAUGAGAAAAAGAAUGACC GGUCAUUCUUUUUCUCAUC 1443 1444 960-978 AUGAGAAAAAGAAUGACCA UGGUCAUUCUUUUUCUCAU 1445 1446 961-979 UGAGAAAAAGAAUGACCAC GUGGUCAUUCUUUUUCUCA 1447 1448 962-980 GAGAAAAAGAAUGACCACA UGUGGUCAUUCUUUUUCUC 1449 1450 963-981 AGAAAAAGAAUGACCACAC GUGUGGUCAUUCUUUUUCU 1451 1452 964-982 GAAAAAGAAUGACCACACC GGUGUGGUCAUUCUUUUUC 1453 1454 965-983 AAAAAGAAUGACCACACCU AGGUGUGGUCAUUCUUUUU 1455 1456 966-984 AAAAGAAUGACCACACCUA UAGGUGUGGUCAUUCUUUU 1457 1458 967-985 AAAGAAUGACCACACCUAU AUAGGUGUGGUCAUUCUUU 1459 1460 968-986 AAGAAUGACCACACCUAUC GAUAGGUGUGGUCAUUCUU 1461 1462 969-987 AGAAUGACCACACCUAUCG CGAUAGGUGUGGUCAUUCU 1463 1464 970-988 GAAUGACCACACCUAUCGA UCGAUAGGUGUGGUCAUUC 1465 1466 971-989 AAUGACCACACCUAUCGAG CUCGAUAGGUGUGGUCAUU 1467 1468 972-990 AUGACCACACCUAUCGAGU ACUCGAUAGGUGUGGUCAU 1469 1470 976-994 CCACACCUAUCGAGUUUUU AAAAACUCGAUAGGUGUGG 1471 1472 977-995 CACACCUAUCGAGUUUUUA UAAAAACUCGAUAGGUGUG 1473 1474 978-996 ACACCUAUCGAGUUUUUAA UUAAAAACUCGAUAGGUGU 1475 1476 979-997 CACCUAUCGAGUUUUUAAA UUUAAAAACUCGAUAGGUG 1477 1478 980-998 ACCUAUCGAGUUUUUAAAA UUUUAAAAACUCGAUAGGU 1479 1480 981-999 CCUAUCGAGUUUUUAAAAC GUUUUAAAAACUCGAUAGG 1481 1482  982-1000 CUAUCGAGUUUUUAAAACU AGUUUUAAAAACUCGAUAG 1483 1484  983-1001 UAUCGAGUUUUUAAAACUG CAGUUUUAAAAACUCGAUA 1485 1486  984-1002 AUCGAGUUUUUAAAACUGU ACAGUUUUAAAAACUCGAU 1487 1488  985-1003 UCGAGUUUUUAAAACUGUG CACAGUUUUAAAAACUCGA 1489 1490  986-1004 CGAGUUUUUAAAACUGUGA UCACAGUUUUAAAAACUCG 1491 1492  987-1005 GAGUUUUUAAAACUGUGAA UUCACAGUUUUAAAAACUC 1493 1494  988-1006 AGUUUUUAAAACUGUGAAC GUUCACAGUUUUAAAAACU 1495 1496  989-1007 GUUUUUAAAACUGUGAACC GGUUCACAGUUUUAAAAAC 1497 1498  990-1008 UUUUUAAAACUGUGAACCG CGGUUCACAGUUUUAAAAA 1499 1500  991-1009 UUUUAAAACUGUGAACCGG CCGGUUCACAGUUUUAAAA 1501 1502  992-1010 UUUAAAACUGUGAACCGGC GCCGGUUCACAGUUUUAAA 1503 1504  993-1011 UUAAAACUGUGAACCGGCG CGCCGGUUCACAGUUUUAA 1505 1506  994-1012 UAAAACUGUGAACCGGCGA UCGCCGGUUCACAGUUUUA 1507 1508  995-1013 AAAACUGUGAACCGGCGAG CUCGCCGGUUCACAGUUUU 1509 1510  996-1014 AAACUGUGAACCGGCGAGC GCUCGCCGGUUCACAGUUU 1511 1512  997-1015 AACUGUGAACCGGCGAGCA UGCUCGCCGGUUCACAGUU 1513 1514  998-1016 ACUGUGAACCGGCGAGCAC GUGCUCGCCGGUUCACAGU 1515 1516  999-1017 CUGUGAACCGGCGAGCACA UGUGCUCGCCGGUUCACAG 1517 1518 1000-1018 UGUGAACCGGCGAGCACAC GUGUGCUCGCCGGUUCACA 1519 1520 1001-1019 GUGAACCGGCGAGCACACA UGUGUGCUCGCCGGUUCAC 1521 1522 1002-1020 UGAACCGGCGAGCACACAU AUGUGUGCUCGCCGGUUCA 1523 1524 1003-1021 GAACCGGCGAGCACACAUC GAUGUGUGCUCGCCGGUUC 1525 1526 1004-1022 AACCGGCGAGCACACAUCU AGAUGUGUGCUCGCCGGUU 1527 1528 1005-1023 ACCGGCGAGCACACAUCUU AAGAUGUGUGCUCGCCGGU 1529 1530 1006-1024 CCGGCGAGCACACAUCUUC GAAGAUGUGUGCUCGCCGG 1531 1532 1007-1025 CGGCGAGCACACAUCUUCC GGAAGAUGUGUGCUCGCCG 1533 1534 1008-1026 GGCGAGCACACAUCUUCCC GGGAAGAUGUGUGCUCGCC 1535 1536 1028-1046 AUGGCAGAUGACUAUUCAG CUGAAUAGUCAUCUGCCAU 1537 1538 1030-1048 GGCAGAUGACUAUUCAGAC GUCUGAAUAGUCAUCUGCC 1539 1540 1031-1049 GCAGAUGACUAUUCAGACU AGUCUGAAUAGUCAUCUGC 1541 1542 1032-1050 CAGAUGACUAUUCAGACUC GAGUCUGAAUAGUCAUCUG 1543 1544 1033-1051 AGAUGACUAUUCAGACUCC GGAGUCUGAAUAGUCAUCU 1545 1546 1034-1052 GAUGACUAUUCAGACUCCC GGGAGUCUGAAUAGUCAUC 1547 1548 1035-1053 AUGACUAUUCAGACUCCCU AGGGAGUCUGAAUAGUCAU 1549 1550 1036-1054 UGACUAUUCAGACUCCCUC GAGGGAGUCUGAAUAGUCA 1551 1552 1037-1055 GACUAUUCAGACUCCCUCA UGAGGGAGUCUGAAUAGUC 1553 1554 1038-1056 ACUAUUCAGACUCCCUCAU AUGAGGGAGUCUGAAUAGU 1555 1556 1039-1057 CUAUUCAGACUCCCUCAUC GAUGAGGGAGUCUGAAUAG 1557 1558 1040-1058 UAUUCAGACUCCCUCAUCA UGAUGAGGGAGUCUGAAUA 1559 1560 1041-1059 AUUCAGACUCCCUCAUCAC GUGAUGAGGGAGUCUGAAU 1561 1562 1042-1060 UUCAGACUCCCUCAUCACC GGUGAUGAGGGAGUCUGAA 1563 1564 1043-1061 UCAGACUCCCUCAUCACCA UGGUGAUGAGGGAGUCUGA 1565 1566 1044-1062 CAGACUCCCUCAUCACCAA UUGGUGAUGAGGGAGUCUG 1567 1568 1045-1063 AGACUCCCUCAUCACCAAA UUUGGUGAUGAGGGAGUCU 1569 1570 1046-1064 GACUCCCUCAUCACCAAAA UUUUGGUGAUGAGGGAGUC 1571 1572 1047-1065 ACUCCCUCAUCACCAAAAA UUUUUGGUGAUGAGGGAGU 1573 1574 1048-1066 CUCCCUCAUCACCAAAAAG CUUUUUGGUGAUGAGGGAG 1575 1576 1049-1067 UCCCUCAUCACCAAAAAGC GCUUUUUGGUGAUGAGGGA 1577 1578 1050-1068 CCCUCAUCACCAAAAAGCA UGCUUUUUGGUGAUGAGGG 1579 1580 1070-1088 GUGUCAGUCUGGUGCAGUA UACUGCACCAGACUGACAC 1581 1582 1071-1089 UGUCAGUCUGGUGCAGUAA UUACUGCACCAGACUGACA 1583 1584 1072-1090 GUCAGUCUGGUGCAGUAAU AUUACUGCACCAGACUGAC 1585 1586 1073-1091 UCAGUCUGGUGCAGUAAUG CAUUACUGCACCAGACUGA 1587 1588 1074-1092 CAGUCUGGUGCAGUAAUGA UCAUUACUGCACCAGACUG 1589 1590 1075-1093 AGUCUGGUGCAGUAAUGAC GUCAUUACUGCACCAGACU 1591 1592 1078-1096 CUGGUGCAGUAAUGACUAC GUAGUCAUUACUGCACCAG 1593 1594 1079-1097 UGGUGCAGUAAUGACUACC GGUAGUCAUUACUGCACCA 1595 1596 1081-1099 GUGCAGUAAUGACUACCUA UAGGUAGUCAUUACUGCAC 1597 1598 1082-1100 UGCAGUAAUGACUACCUAG CUAGGUAGUCAUUACUGCA 1599 1600 1083-1101 GCAGUAAUGACUACCUAGG CCUAGGUAGUCAUUACUGC 1601 1602 1084-1102 CAGUAAUGACUACCUAGGA UCCUAGGUAGUCAUUACUG 1603 1604 1085-1103 AGUAAUGACUACCUAGGAA UUCCUAGGUAGUCAUUACU 1605 1606 1086-1104 GUAAUGACUACCUAGGAAU AUUCCUAGGUAGUCAUUAC 1607 1608 1087-1105 UAAUGACUACCUAGGAAUG CAUUCCUAGGUAGUCAUUA 1609 1610 1088-1106 AAUGACUACCUAGGAAUGA UCAUUCCUAGGUAGUCAUU 1611 1612 1089-1107 AUGACUACCUAGGAAUGAG CUCAUUCCUAGGUAGUCAU 1613 1614 1090-1108 UGACUACCUAGGAAUGAGU ACUCAUUCCUAGGUAGUCA 1615 1616 1091-1109 GACUACCUAGGAAUGAGUC GACUCAUUCCUAGGUAGUC 1617 1618 1092-1110 ACUACCUAGGAAUGAGUCG CGACUCAUUCCUAGGUAGU 1619 1620 1093-1111 CUACCUAGGAAUGAGUCGC GCGACUCAUUCCUAGGUAG 1621 1622 1094-1112 UACCUAGGAAUGAGUCGCC GGCGACUCAUUCCUAGGUA 1623 1624 1095-1113 ACCUAGGAAUGAGUCGCCA UGGCGACUCAUUCCUAGGU 1625 1626 1096-1114 CCUAGGAAUGAGUCGCCAC GUGGCGACUCAUUCCUAGG 1627 1628 1097-1115 CUAGGAAUGAGUCGCCACC GGUGGCGACUCAUUCCUAG 1629 1630 1098-1116 UAGGAAUGAGUCGCCACCC GGGUGGCGACUCAUUCCUA 1631 1632 1099-1117 AGGAAUGAGUCGCCACCCA UGGGUGGCGACUCAUUCCU 1633 1634 1100-1118 GGAAUGAGUCGCCACCCAC GUGGGUGGCGACUCAUUCC 1635 1636 1101-1119 GAAUGAGUCGCCACCCACG CGUGGGUGGCGACUCAUUC 1637 1638 1102-1120 AAUGAGUCGCCACCCACGG CCGUGGGUGGCGACUCAUU 1639 1640 1103-1121 AUGAGUCGCCACCCACGGG CCCGUGGGUGGCGACUCAU 1641 1642 1104-1122 UGAGUCGCCACCCACGGGU ACCCGUGGGUGGCGACUCA 1643 1644 1105-1123 GAGUCGCCACCCACGGGUG CACCCGUGGGUGGCGACUC 1645 1646 1106-1124 AGUCGCCACCCACGGGUGU ACACCCGUGGGUGGCGACU 1647 1648 1107-1125 GUCGCCACCCACGGGUGUG CACACCCGUGGGUGGCGAC 1649 1650 1108-1126 UCGCCACCCACGGGUGUGU ACACACCCGUGGGUGGCGA 1651 1652 1109-1127 CGCCACCCACGGGUGUGUG CACACACCCGUGGGUGGCG 1653 1654 1110-1128 GCCACCCACGGGUGUGUGG CCACACACCCGUGGGUGGC 1655 1656 1111-1129 CCACCCACGGGUGUGUGGG CCCACACACCCGUGGGUGG 1657 1658 1112-1130 CACCCACGGGUGUGUGGGG CCCCACACACCCGUGGGUG 1659 1660 1113-1131 ACCCACGGGUGUGUGGGGC GCCCCACACACCCGUGGGU 1661 1662 1114-1132 CCCACGGGUGUGUGGGGCA UGCCCCACACACCCGUGGG 1663 1664 1115-1133 CCACGGGUGUGUGGGGCAG CUGCCCCACACACCCGUGG 1665 1666 1116-1134 CACGGGUGUGUGGGGCAGU ACUGCCCCACACACCCGUG 1667 1668 1117-1135 ACGGGUGUGUGGGGCAGUU AACUGCCCCACACACCCGU 1669 1670 1118-1136 CGGGUGUGUGGGGCAGUUA UAACUGCCCCACACACCCG 1671 1672 1119-1137 GGGUGUGUGGGGCAGUUAU AUAACUGCCCCACACACCC 1673 1674 1120-1138 GGUGUGUGGGGCAGUUAUG CAUAACUGCCCCACACACC 1675 1676 1121-1139 GUGUGUGGGGCAGUUAUGG CCAUAACUGCCCCACACAC 1677 1678 1122-1140 UGUGUGGGGCAGUUAUGGA UCCAUAACUGCCCCACACA 1679 1680 1123-1141 GUGUGGGGCAGUUAUGGAC GUCCAUAACUGCCCCACAC 1681 1682 1125-1143 GUGGGGCAGUUAUGGACAC GUGUCCAUAACUGCCCCAC 1683 1684 1126-1144 UGGGGCAGUUAUGGACACU AGUGUCCAUAACUGCCCCA 1685 1686 1128-1146 GGGCAGUUAUGGACACUUU AAAGUGUCCAUAACUGCCC 1687 1688 1129-1147 GGCAGUUAUGGACACUUUG CAAAGUGUCCAUAACUGCC 1689 1690 1130-1148 GCAGUUAUGGACACUUUGA UCAAAGUGUCCAUAACUGC 1691 1692 1131-1149 CAGUUAUGGACACUUUGAA UUCAAAGUGUCCAUAACUG 1693 1694 1132-1150 AGUUAUGGACACUUUGAAA UUUCAAAGUGUCCAUAACU 1695 1696 1133-1151 GUUAUGGACACUUUGAAAC GUUUCAAAGUGUCCAUAAC 1697 1698 1134-1152 UUAUGGACACUUUGAAACA UGUUUCAAAGUGUCCAUAA 1699 1700 1135-1153 UAUGGACACUUUGAAACAA UUGUUUCAAAGUGUCCAUA 1701 1702 1136-1154 AUGGACACUUUGAAACAAC GUUGUUUCAAAGUGUCCAU 1703 1704 1139-1157 GACACUUUGAAACAACAUG CAUGUUGUUUCAAAGUGUC 1705 1706 1140-1158 ACACUUUGAAACAACAUGG CCAUGUUGUUUCAAAGUGU 1707 1708 1141-1159 CACUUUGAAACAACAUGGU ACCAUGUUGUUUCAAAGUG 1709 1710 1142-1160 ACUUUGAAACAACAUGGUG CACCAUGUUGUUUCAAAGU 1711 1712 1143-1161 CUUUGAAACAACAUGGUGC GCACCAUGUUGUUUCAAAG 1713 1714 1144-1162 UUUGAAACAACAUGGUGCU AGCACCAUGUUGUUUCAAA 1715 1716 1145-1163 UUGAAACAACAUGGUGCUG CAGCACCAUGUUGUUUCAA 1717 1718 1146-1164 UGAAACAACAUGGUGCUGG CCAGCACCAUGUUGUUUCA 1719 1720 1147-1165 GAAACAACAUGGUGCUGGG CCCAGCACCAUGUUGUUUC 1721 1722 1148-1166 AAACAACAUGGUGCUGGGG CCCCAGCACCAUGUUGUUU 1723 1724 1149-1167 AACAACAUGGUGCUGGGGC GCCCCAGCACCAUGUUGUU 1725 1726 1150-1168 ACAACAUGGUGCUGGGGCA UGCCCCAGCACCAUGUUGU 1727 1728 1151-1169 CAACAUGGUGCUGGGGCAG CUGCCCCAGCACCAUGUUG 1729 1730 1152-1170 AACAUGGUGCUGGGGCAGG CCUGCCCCAGCACCAUGUU 1731 1732 1153-1171 ACAUGGUGCUGGGGCAGGU ACCUGCCCCAGCACCAUGU 1733 1734 1154-1172 CAUGGUGCUGGGGCAGGUG CACCUGCCCCAGCACCAUG 1735 1736 1155-1173 AUGGUGCUGGGGCAGGUGG CCACCUGCCCCAGCACCAU 1737 1738 1156-1174 UGGUGCUGGGGCAGGUGGU ACCACCUGCCCCAGCACCA 1739 1740 1157-1175 GGUGCUGGGGCAGGUGGUA UACCACCUGCCCCAGCACC 1741 1742 1158-1176 GUGCUGGGGCAGGUGGUAC GUACCACCUGCCCCAGCAC 1743 1744 1159-1177 UGCUGGGGCAGGUGGUACU AGUACCACCUGCCCCAGCA 1745 1746 1160-1178 GCUGGGGCAGGUGGUACUA UAGUACCACCUGCCCCAGC 1747 1748 1161-1179 CUGGGGCAGGUGGUACUAG CUAGUACCACCUGCCCCAG 1749 1750 1162-1180 UGGGGCAGGUGGUACUAGA UCUAGUACCACCUGCCCCA 1751 1752 1166-1184 GCAGGUGGUACUAGAAAUA UAUUUCUAGUACCACCUGC 1753 1754 1167-1185 CAGGUGGUACUAGAAAUAU AUAUUUCUAGUACCACCUG 1755 1756 1168-1186 AGGUGGUACUAGAAAUAUU AAUAUUUCUAGUACCACCU 1757 1758 1169-1187 GGUGGUACUAGAAAUAUUU AAAUAUUUCUAGUACCACC 1759 1760 1170-1188 GUGGUACUAGAAAUAUUUC GAAAUAUUUCUAGUACCAC 1761 1762 1171-1189 UGGUACUAGAAAUAUUUCU AGAAAUAUUUCUAGUACCA 1763 1764 1172-1190 GGUACUAGAAAUAUUUCUG CAGAAAUAUUUCUAGUACC 1765 1766 1173-1191 GUACUAGAAAUAUUUCUGG CCAGAAAUAUUUCUAGUAC 1767 1768 1174-1192 UACUAGAAAUAUUUCUGGA UCCAGAAAUAUUUCUAGUA 1769 1770 1175-1193 ACUAGAAAUAUUUCUGGAA UUCCAGAAAUAUUUCUAGU 1771 1772 1176-1194 CUAGAAAUAUUUCUGGAAC GUUCCAGAAAUAUUUCUAG 1773 1774 1177-1195 UAGAAAUAUUUCUGGAACU AGUUCCAGAAAUAUUUCUA 1775 1776 1178-1196 AGAAAUAUUUCUGGAACUA UAGUUCCAGAAAUAUUUCU 1777 1778 1179-1197 GAAAUAUUUCUGGAACUAG CUAGUUCCAGAAAUAUUUC 1779 1780 1180-1198 AAAUAUUUCUGGAACUAGU ACUAGUUCCAGAAAUAUUU 1781 1782 1181-1199 AAUAUUUCUGGAACUAGUA UACUAGUUCCAGAAAUAUU 1783 1784 1183-1201 UAUUUCUGGAACUAGUAAA UUUACUAGUUCCAGAAAUA 1785 1786 1186-1204 UUCUGGAACUAGUAAAUUC GAAUUUACUAGUUCCAGAA 1787 1788 1187-1205 UCUGGAACUAGUAAAUUCC GGAAUUUACUAGUUCCAGA 1789 1790 1189-1207 UGGAACUAGUAAAUUCCAU AUGGAAUUUACUAGUUCCA 1791 1792 1190-1208 GGAACUAGUAAAUUCCAUG CAUGGAAUUUACUAGUUCC 1793 1794 1192-1210 AACUAGUAAAUUCCAUGUG CACAUGGAAUUUACUAGUU 1795 1796 1193-1211 ACUAGUAAAUUCCAUGUGG CCACAUGGAAUUUACUAGU 1797 1798 1194-1212 CUAGUAAAUUCCAUGUGGA UCCACAUGGAAUUUACUAG 1799 1800 1195-1213 UAGUAAAUUCCAUGUGGAC GUCCACAUGGAAUUUACUA 1801 1802 1196-1214 AGUAAAUUCCAUGUGGACU AGUCCACAUGGAAUUUACU 1803 1804 1197-1215 GUAAAUUCCAUGUGGACUU AAGUCCACAUGGAAUUUAC 1805 1806 1198-1216 UAAAUUCCAUGUGGACUUA UAAGUCCACAUGGAAUUUA 1807 1808 1199-1217 AAAUUCCAUGUGGACUUAG CUAAGUCCACAUGGAAUUU 1809 1810 1200-1218 AAUUCCAUGUGGACUUAGA UCUAAGUCCACAUGGAAUU 1811 1812 1201-1219 AUUCCAUGUGGACUUAGAG CUCUAAGUCCACAUGGAAU 1813 1814 1202-1220 UUCCAUGUGGACUUAGAGC GCUCUAAGUCCACAUGGAA 1815 1816 1222-1240 GGAGCUGGCAGACCUCCAU AUGGAGGUCUGCCAGCUCC 1817 1818 1223-1241 GAGCUGGCAGACCUCCAUG CAUGGAGGUCUGCCAGCUC 1819 1820 1224-1242 AGCUGGCAGACCUCCAUGG CCAUGGAGGUCUGCCAGCU 1821 1822 1225-1243 GCUGGCAGACCUCCAUGGG CCCAUGGAGGUCUGCCAGC 1823 1824 1226-1244 CUGGCAGACCUCCAUGGGA UCCCAUGGAGGUCUGCCAG 1825 1826 1227-1245 UGGCAGACCUCCAUGGGAA UUCCCAUGGAGGUCUGCCA 1827 1828 1228-1246 GGCAGACCUCCAUGGGAAA UUUCCCAUGGAGGUCUGCC 1829 1830 1229-1247 GCAGACCUCCAUGGGAAAG CUUUCCCAUGGAGGUCUGC 1831 1832 1230-1248 CAGACCUCCAUGGGAAAGA UCUUUCCCAUGGAGGUCUG 1833 1834 1231-1249 AGACCUCCAUGGGAAAGAU AUCUUUCCCAUGGAGGUCU 1835 1836 1232-1250 GACCUCCAUGGGAAAGAUG CAUCUUUCCCAUGGAGGUC 1837 1838 1233-1251 ACCUCCAUGGGAAAGAUGC GCAUCUUUCCCAUGGAGGU 1839 1840 1254-1272 CACUCUUGUUUUCCUCGUG CACGAGGAAAACAAGAGUG 1841 1842 1255-1273 ACUCUUGUUUUCCUCGUGC GCACGAGGAAAACAAGAGU 1843 1844 1256-1274 CUCUUGUUUUCCUCGUGCU AGCACGAGGAAAACAAGAG 1845 1846 1257-1275 UCUUGUUUUCCUCGUGCUU AAGCACGAGGAAAACAAGA 1847 1848 1259-1277 UUGUUUUCCUCGUGCUUUG CAAAGCACGAGGAAAACAA 1849 1850 1260-1278 UGUUUUCCUCGUGCUUUGU ACAAAGCACGAGGAAAACA 1851 1852 1261-1279 GUUUUCCUCGUGCUUUGUG CACAAAGCACGAGGAAAAC 1853 1854 1262-1280 UUUUCCUCGUGCUUUGUGG CCACAAAGCACGAGGAAAA 1855 1856 1263-1281 UUUCCUCGUGCUUUGUGGC GCCACAAAGCACGAGGAAA 1857 1858 1264-1282 UUCCUCGUGCUUUGUGGCC GGCCACAAAGCACGAGGAA 1859 1860 1265-1283 UCCUCGUGCUUUGUGGCCA UGGCCACAAAGCACGAGGA 1861 1862 1266-1284 CCUCGUGCUUUGUGGCCAA UUGGCCACAAAGCACGAGG 1863 1864 1267-1285 CUCGUGCUUUGUGGCCAAU AUUGGCCACAAAGCACGAG 1865 1866 1268-1286 UCGUGCUUUGUGGCCAAUG CAUUGGCCACAAAGCACGA 1867 1868 1269-1287 CGUGCUUUGUGGCCAAUGA UCAUUGGCCACAAAGCACG 1869 1870 1270-1288 GUGCUUUGUGGCCAAUGAC GUCAUUGGCCACAAAGCAC 1871 1872 1271-1289 UGCUUUGUGGCCAAUGACU AGUCAUUGGCCACAAAGCA 1873 1874 1272-1290 GCUUUGUGGCCAAUGACUC GAGUCAUUGGCCACAAAGC 1875 1876 1273-1291 CUUUGUGGCCAAUGACUCA UGAGUCAUUGGCCACAAAG 1877 1878 1274-1292 UUUGUGGCCAAUGACUCAA UUGAGUCAUUGGCCACAAA 1879 1880 1275-1293 UUGUGGCCAAUGACUCAAC GUUGAGUCAUUGGCCACAA 1881 1882 1276-1294 UGUGGCCAAUGACUCAACC GGUUGAGUCAUUGGCCACA 1883 1884 1277-1295 GUGGCCAAUGACUCAACCC GGGUUGAGUCAUUGGCCAC 1885 1886 1278-1296 UGGCCAAUGACUCAACCCU AGGGUUGAGUCAUUGGCCA 1887 1888 1279-1297 GGCCAAUGACUCAACCCUC GAGGGUUGAGUCAUUGGCC 1889 1890 1280-1298 GCCAAUGACUCAACCCUCU AGAGGGUUGAGUCAUUGGC 1891 1892 1281-1299 CCAAUGACUCAACCCUCUU AAGAGGGUUGAGUCAUUGG 1893 1894 1282-1300 CAAUGACUCAACCCUCUUC GAAGAGGGUUGAGUCAUUG 1895 1896 1283-1301 AAUGACUCAACCCUCUUCA UGAAGAGGGUUGAGUCAUU 1897 1898 1284-1302 AUGACUCAACCCUCUUCAC GUGAAGAGGGUUGAGUCAU 1899 1900 1285-1303 UGACUCAACCCUCUUCACC GGUGAAGAGGGUUGAGUCA 1901 1902 1286-1304 GACUCAACCCUCUUCACCC GGGUGAAGAGGGUUGAGUC 1903 1904 1287-1305 ACUCAACCCUCUUCACCCU AGGGUGAAGAGGGUUGAGU 1905 1906 1288-1306 CUCAACCCUCUUCACCCUG CAGGGUGAAGAGGGUUGAG 1907 1908 1289-1307 UCAACCCUCUUCACCCUGG CCAGGGUGAAGAGGGUUGA 1909 1910 1290-1308 CAACCCUCUUCACCCUGGC GCCAGGGUGAAGAGGGUUG 1911 1912 1291-1309 AACCCUCUUCACCCUGGCU AGCCAGGGUGAAGAGGGUU 1913 1914 1292-1310 ACCCUCUUCACCCUGGCUA UAGCCAGGGUGAAGAGGGU 1915 1916 1293-1311 CCCUCUUCACCCUGGCUAA UUAGCCAGGGUGAAGAGGG 1917 1918 1294-1312 CCUCUUCACCCUGGCUAAG CUUAGCCAGGGUGAAGAGG 1919 1920 1297-1315 CUUCACCCUGGCUAAGAUG CAUCUUAGCCAGGGUGAAG 1921 1922 1298-1316 UUCACCCUGGCUAAGAUGA UCAUCUUAGCCAGGGUGAA 1923 1924 1300-1318 CACCCUGGCUAAGAUGAUG CAUCAUCUUAGCCAGGGUG 1925 1926 1301-1319 ACCCUGGCUAAGAUGAUGC GCAUCAUCUUAGCCAGGGU 1927 1928 1302-1320 CCCUGGCUAAGAUGAUGCC GGCAUCAUCUUAGCCAGGG 1929 1930 1303-1321 CCUGGCUAAGAUGAUGCCA UGGCAUCAUCUUAGCCAGG 1931 1932 1304-1322 CUGGCUAAGAUGAUGCCAG CUGGCAUCAUCUUAGCCAG 1933 1934 1305-1323 UGGCUAAGAUGAUGCCAGG CCUGGCAUCAUCUUAGCCA 1935 1936 1306-1324 GGCUAAGAUGAUGCCAGGC GCCUGGCAUCAUCUUAGCC 1937 1938 1307-1325 GCUAAGAUGAUGCCAGGCU AGCCUGGCAUCAUCUUAGC 1939 1940 1308-1326 CUAAGAUGAUGCCAGGCUG CAGCCUGGCAUCAUCUUAG 1941 1942 1309-1327 UAAGAUGAUGCCAGGCUGU ACAGCCUGGCAUCAUCUUA 1943 1944 1310-1328 AAGAUGAUGCCAGGCUGUG CACAGCCUGGCAUCAUCUU 1945 1946 1311-1329 AGAUGAUGCCAGGCUGUGA UCACAGCCUGGCAUCAUCU 1947 1948 1312-1330 GAUGAUGCCAGGCUGUGAG CUCACAGCCUGGCAUCAUC 1949 1950 1313-1331 AUGAUGCCAGGCUGUGAGA UCUCACAGCCUGGCAUCAU 1951 1952 1314-1332 UGAUGCCAGGCUGUGAGAU AUCUCACAGCCUGGCAUCA 1953 1954 1316-1334 AUGCCAGGCUGUGAGAUUU AAAUCUCACAGCCUGGCAU 1955 1956 1317-1335 UGCCAGGCUGUGAGAUUUA UAAAUCUCACAGCCUGGCA 1957 1958 1318-1336 GCCAGGCUGUGAGAUUUAC GUAAAUCUCACAGCCUGGC 1959 1960 1319-1337 CCAGGCUGUGAGAUUUACU AGUAAAUCUCACAGCCUGG 1961 1962 1320-1338 CAGGCUGUGAGAUUUACUC GAGUAAAUCUCACAGCCUG 1963 1964 1321-1339 AGGCUGUGAGAUUUACUCU AGAGUAAAUCUCACAGCCU 1965 1966 1322-1340 GGCUGUGAGAUUUACUCUG CAGAGUAAAUCUCACAGCC 1967 1968 1323-1341 GCUGUGAGAUUUACUCUGA UCAGAGUAAAUCUCACAGC 1969 1970 1326-1344 GUGAGAUUUACUCUGAUUC GAAUCAGAGUAAAUCUCAC 1971 1972 1327-1345 UGAGAUUUACUCUGAUUCU AGAAUCAGAGUAAAUCUCA 1973 1974 1328-1346 GAGAUUUACUCUGAUUCUG CAGAAUCAGAGUAAAUCUC 1975 1976 1329-1347 AGAUUUACUCUGAUUCUGG CCAGAAUCAGAGUAAAUCU 1977 1978 1330-1348 GAUUUACUCUGAUUCUGGG CCCAGAAUCAGAGUAAAUC 1979 1980 1331-1349 AUUUACUCUGAUUCUGGGA UCCCAGAAUCAGAGUAAAU 1981 1982 1332-1350 UUUACUCUGAUUCUGGGAA UUCCCAGAAUCAGAGUAAA 1983 1984 1333-1351 UUACUCUGAUUCUGGGAAC GUUCCCAGAAUCAGAGUAA 1985 1986 1334-1352 UACUCUGAUUCUGGGAACC GGUUCCCAGAAUCAGAGUA 1987 1988 1335-1353 ACUCUGAUUCUGGGAACCA UGGUUCCCAGAAUCAGAGU 1989 1990 1336-1354 CUCUGAUUCUGGGAACCAU AUGGUUCCCAGAAUCAGAG 1991 1992 1337-1355 UCUGAUUCUGGGAACCAUG CAUGGUUCCCAGAAUCAGA 1993 1994 1338-1356 CUGAUUCUGGGAACCAUGC GCAUGGUUCCCAGAAUCAG 1995 1996 1339-1357 UGAUUCUGGGAACCAUGCC GGCAUGGUUCCCAGAAUCA 1997 1998 1340-1358 GAUUCUGGGAACCAUGCCU AGGCAUGGUUCCCAGAAUC 1999 2000 1341-1359 AUUCUGGGAACCAUGCCUC GAGGCAUGGUUCCCAGAAU 2001 2002 1342-1360 UUCUGGGAACCAUGCCUCC GGAGGCAUGGUUCCCAGAA 2003 2004 1343-1361 UCUGGGAACCAUGCCUCCA UGGAGGCAUGGUUCCCAGA 2005 2006 1344-1362 CUGGGAACCAUGCCUCCAU AUGGAGGCAUGGUUCCCAG 2007 2008 1345-1363 UGGGAACCAUGCCUCCAUG CAUGGAGGCAUGGUUCCCA 2009 2010 1346-1364 GGGAACCAUGCCUCCAUGA UCAUGGAGGCAUGGUUCCC 2011 2012 1348-1366 GAACCAUGCCUCCAUGAUC GAUCAUGGAGGCAUGGUUC 2013 2014 1349-1367 AACCAUGCCUCCAUGAUCC GGAUCAUGGAGGCAUGGUU 2015 2016 1350-1368 ACCAUGCCUCCAUGAUCCA UGGAUCAUGGAGGCAUGGU 2017 2018 1351-1369 CCAUGCCUCCAUGAUCCAA UUGGAUCAUGGAGGCAUGG 2019 2020 1352-1370 CAUGCCUCCAUGAUCCAAG CUUGGAUCAUGGAGGCAUG 2021 2022 1353-1371 AUGCCUCCAUGAUCCAAGG CCUUGGAUCAUGGAGGCAU 2023 2024 1354-1372 UGCCUCCAUGAUCCAAGGG CCCUUGGAUCAUGGAGGCA 2025 2026 1358-1376 UCCAUGAUCCAAGGGAUUC GAAUCCCUUGGAUCAUGGA 2027 2028 1359-1377 CCAUGAUCCAAGGGAUUCG CGAAUCCCUUGGAUCAUGG 2029 2030 1360-1378 CAUGAUCCAAGGGAUUCGA UCGAAUCCCUUGGAUCAUG 2031 2032 1361-1379 AUGAUCCAAGGGAUUCGAA UUCGAAUCCCUUGGAUCAU 2033 2034 1362-1380 UGAUCCAAGGGAUUCGAAA UUUCGAAUCCCUUGGAUCA 2035 2036 1363-1381 GAUCCAAGGGAUUCGAAAC GUUUCGAAUCCCUUGGAUC 2037 2038 1365-1383 UCCAAGGGAUUCGAAACAG CUGUUUCGAAUCCCUUGGA 2039 2040 1366-1384 CCAAGGGAUUCGAAACAGC GCUGUUUCGAAUCCCUUGG 2041 2042 1367-1385 CAAGGGAUUCGAAACAGCC GGCUGUUUCGAAUCCCUUG 2043 2044 1368-1386 AAGGGAUUCGAAACAGCCG CGGCUGUUUCGAAUCCCUU 2045 2046 1369-1387 AGGGAUUCGAAACAGCCGA UCGGCUGUUUCGAAUCCCU 2047 2048 1370-1388 GGGAUUCGAAACAGCCGAG CUCGGCUGUUUCGAAUCCC 2049 2050 1371-1389 GGAUUCGAAACAGCCGAGU ACUCGGCUGUUUCGAAUCC 2051 2052 1372-1390 GAUUCGAAACAGCCGAGUG CACUCGGCUGUUUCGAAUC 2053 2054 1373-1391 AUUCGAAACAGCCGAGUGC GCACUCGGCUGUUUCGAAU 2055 2056 1374-1392 UUCGAAACAGCCGAGUGCC GGCACUCGGCUGUUUCGAA 2057 2058 1375-1393 UCGAAACAGCCGAGUGCCA UGGCACUCGGCUGUUUCGA 2059 2060 1376-1394 CGAAACAGCCGAGUGCCAA UUGGCACUCGGCUGUUUCG 2061 2062 1377-1395 GAAACAGCCGAGUGCCAAA UUUGGCACUCGGCUGUUUC 2063 2064 1378-1396 AAACAGCCGAGUGCCAAAG CUUUGGCACUCGGCUGUUU 2065 2066 1379-1397 AACAGCCGAGUGCCAAAGU ACUUUGGCACUCGGCUGUU 2067 2068 1380-1398 ACAGCCGAGUGCCAAAGUA UACUUUGGCACUCGGCUGU 2069 2070 1381-1399 CAGCCGAGUGCCAAAGUAC GUACUUUGGCACUCGGCUG 2071 2072 1383-1401 GCCGAGUGCCAAAGUACAU AUGUACUUUGGCACUCGGC 2073 2074 1384-1402 CCGAGUGCCAAAGUACAUC GAUGUACUUUGGCACUCGG 2075 2076 1385-1403 CGAGUGCCAAAGUACAUCU AGAUGUACUUUGGCACUCG 2077 2078 1386-1404 GAGUGCCAAAGUACAUCUU AAGAUGUACUUUGGCACUC 2079 2080 1387-1405 AGUGCCAAAGUACAUCUUC GAAGAUGUACUUUGGCACU 2081 2082 1388-1406 GUGCCAAAGUACAUCUUCC GGAAGAUGUACUUUGGCAC 2083 2084 1389-1407 UGCCAAAGUACAUCUUCCG CGGAAGAUGUACUUUGGCA 2085 2086 1390-1408 GCCAAAGUACAUCUUCCGC GCGGAAGAUGUACUUUGGC 2087 2088 1391-1409 CCAAAGUACAUCUUCCGCC GGCGGAAGAUGUACUUUGG 2089 2090 1392-1410 CAAAGUACAUCUUCCGCCA UGGCGGAAGAUGUACUUUG 2091 2092 1393-1411 AAAGUACAUCUUCCGCCAC GUGGCGGAAGAUGUACUUU 2093 2094 1394-1412 AAGUACAUCUUCCGCCACA UGUGGCGGAAGAUGUACUU 2095 2096 1395-1413 AGUACAUCUUCCGCCACAA UUGUGGCGGAAGAUGUACU 2097 2098 1396-1414 GUACAUCUUCCGCCACAAU AUUGUGGCGGAAGAUGUAC 2099 2100 1397-1415 UACAUCUUCCGCCACAAUG CAUUGUGGCGGAAGAUGUA 2101 2102 1398-1416 ACAUCUUCCGCCACAAUGA UCAUUGUGGCGGAAGAUGU 2103 2104 1399-1417 CAUCUUCCGCCACAAUGAU AUCAUUGUGGCGGAAGAUG 2105 2106 1400-1418 AUCUUCCGCCACAAUGAUG CAUCAUUGUGGCGGAAGAU 2107 2108 1401-1419 UCUUCCGCCACAAUGAUGU ACAUCAUUGUGGCGGAAGA 2109 2110 1402-1420 CUUCCGCCACAAUGAUGUC GACAUCAUUGUGGCGGAAG 2111 2112 1403-1421 UUCCGCCACAAUGAUGUCA UGACAUCAUUGUGGCGGAA 2113 2114 1404-1422 UCCGCCACAAUGAUGUCAG CUGACAUCAUUGUGGCGGA 2115 2116 1405-1423 CCGCCACAAUGAUGUCAGC GCUGACAUCAUUGUGGCGG 2117 2118 1406-1424 CGCCACAAUGAUGUCAGCC GGCUGACAUCAUUGUGGCG 2119 2120 1407-1425 GCCACAAUGAUGUCAGCCA UGGCUGACAUCAUUGUGGC 2121 2122 1427-1445 CUCAGAGAACUGCUGCAAA UUUGCAGCAGUUCUCUGAG 2123 2124 1428-1446 UCAGAGAACUGCUGCAAAG CUUUGCAGCAGUUCUCUGA 2125 2126 1429-1447 CAGAGAACUGCUGCAAAGA UCUUUGCAGCAGUUCUCUG 2127 2128 1430-1448 AGAGAACUGCUGCAAAGAU AUCUUUGCAGCAGUUCUCU 2129 2130 1431-1449 GAGAACUGCUGCAAAGAUC GAUCUUUGCAGCAGUUCUC 2131 2132 1432-1450 AGAACUGCUGCAAAGAUCU AGAUCUUUGCAGCAGUUCU 2133 2134 1433-1451 GAACUGCUGCAAAGAUCUG CAGAUCUUUGCAGCAGUUC 2135 2136 1434-1452 AACUGCUGCAAAGAUCUGA UCAGAUCUUUGCAGCAGUU 2137 2138 1435-1453 ACUGCUGCAAAGAUCUGAC GUCAGAUCUUUGCAGCAGU 2139 2140 1436-1454 CUGCUGCAAAGAUCUGACC GGUCAGAUCUUUGCAGCAG 2141 2142 1437-1455 UGCUGCAAAGAUCUGACCC GGGUCAGAUCUUUGCAGCA 2143 2144 1457-1475 UCAGUCCCCAAGAUUGUGG CCACAAUCUUGGGGACUGA 2145 2146 1458-1476 CAGUCCCCAAGAUUGUGGC GCCACAAUCUUGGGGACUG 2147 2148 1459-1477 AGUCCCCAAGAUUGUGGCA UGCCACAAUCUUGGGGACU 2149 2150 1461-1479 UCCCCAAGAUUGUGGCAUU AAUGCCACAAUCUUGGGGA 2151 2152 1462-1480 CCCCAAGAUUGUGGCAUUU AAAUGCCACAAUCUUGGGG 2153 2154 1463-1481 CCCAAGAUUGUGGCAUUUG CAAAUGCCACAAUCUUGGG 2155 2156 1464-1482 CCAAGAUUGUGGCAUUUGA UCAAAUGCCACAAUCUUGG 2157 2158 1465-1483 CAAGAUUGUGGCAUUUGAA UUCAAAUGCCACAAUCUUG 2159 2160 1466-1484 AAGAUUGUGGCAUUUGAAA UUUCAAAUGCCACAAUCUU 2161 2162 1467-1485 AGAUUGUGGCAUUUGAAAC GUUUCAAAUGCCACAAUCU 2163 2164 1468-1486 GAUUGUGGCAUUUGAAACU AGUUUCAAAUGCCACAAUC 2165 2166 1469-1487 AUUGUGGCAUUUGAAACUG CAGUUUCAAAUGCCACAAU 2167 2168 1470-1488 UUGUGGCAUUUGAAACUGU ACAGUUUCAAAUGCCACAA 2169 2170 1471-1489 UGUGGCAUUUGAAACUGUC GACAGUUUCAAAUGCCACA 2171 2172 1472-1490 GUGGCAUUUGAAACUGUCC GGACAGUUUCAAAUGCCAC 2173 2174 1473-1491 UGGCAUUUGAAACUGUCCA UGGACAGUUUCAAAUGCCA 2175 2176 1474-1492 GGCAUUUGAAACUGUCCAU AUGGACAGUUUCAAAUGCC 2177 2178 1475-1493 GCAUUUGAAACUGUCCAUU AAUGGACAGUUUCAAAUGC 2179 2180 1476-1494 CAUUUGAAACUGUCCAUUC GAAUGGACAGUUUCAAAUG 2181 2182 1477-1495 AUUUGAAACUGUCCAUUCA UGAAUGGACAGUUUCAAAU 2183 2184 1479-1497 UUGAAACUGUCCAUUCAAU AUUGAAUGGACAGUUUCAA 2185 2186 1480-1498 UGAAACUGUCCAUUCAAUG CAUUGAAUGGACAGUUUCA 2187 2188 1481-1499 GAAACUGUCCAUUCAAUGG CCAUUGAAUGGACAGUUUC 2189 2190 1482-1500 AAACUGUCCAUUCAAUGGA UCCAUUGAAUGGACAGUUU 2191 2192 1483-1501 AACUGUCCAUUCAAUGGAU AUCCAUUGAAUGGACAGUU 2193 2194 1484-1502 ACUGUCCAUUCAAUGGAUG CAUCCAUUGAAUGGACAGU 2195 2196 1485-1503 CUGUCCAUUCAAUGGAUGG CCAUCCAUUGAAUGGACAG 2197 2198 1486-1504 UGUCCAUUCAAUGGAUGGG CCCAUCCAUUGAAUGGACA 2199 2200 1487-1505 GUCCAUUCAAUGGAUGGGG CCCCAUCCAUUGAAUGGAC 2201 2202 1488-1506 UCCAUUCAAUGGAUGGGGC GCCCCAUCCAUUGAAUGGA 2203 2204 1508-1526 GUGUGCCCACUGGAAGAGC GCUCUUCCAGUGGGCACAC 2205 2206 1509-1527 UGUGCCCACUGGAAGAGCU AGCUCUUCCAGUGGGCACA 2207 2208 1510-1528 GUGCCCACUGGAAGAGCUG CAGCUCUUCCAGUGGGCAC 2209 2210 1511-1529 UGCCCACUGGAAGAGCUGU ACAGCUCUUCCAGUGGGCA 2211 2212 1512-1530 GCCCACUGGAAGAGCUGUG CACAGCUCUUCCAGUGGGC 2213 2214 1513-1531 CCCACUGGAAGAGCUGUGU ACACAGCUCUUCCAGUGGG 2215 2216 1514-1532 CCACUGGAAGAGCUGUGUG CACACAGCUCUUCCAGUGG 2217 2218 1515-1533 CACUGGAAGAGCUGUGUGA UCACACAGCUCUUCCAGUG 2219 2220 1516-1534 ACUGGAAGAGCUGUGUGAU AUCACACAGCUCUUCCAGU 2221 2222 1517-1535 CUGGAAGAGCUGUGUGAUG CAUCACACAGCUCUUCCAG 2223 2224 1518-1536 UGGAAGAGCUGUGUGAUGU ACAUCACACAGCUCUUCCA 2225 2226 1519-1537 GGAAGAGCUGUGUGAUGUG CACAUCACACAGCUCUUCC 2227 2228 1520-1538 GAAGAGCUGUGUGAUGUGG CCACAUCACACAGCUCUUC 2229 2230 1521-1539 AAGAGCUGUGUGAUGUGGC GCCACAUCACACAGCUCUU 2231 2232 1522-1540 AGAGCUGUGUGAUGUGGCC GGCCACAUCACACAGCUCU 2233 2234 1523-1541 GAGCUGUGUGAUGUGGCCC GGGCCACAUCACACAGCUC 2235 2236 1524-1542 AGCUGUGUGAUGUGGCCCA UGGGCCACAUCACACAGCU 2237 2238 1525-1543 GCUGUGUGAUGUGGCCCAU AUGGGCCACAUCACACAGC 2239 2240 1526-1544 CUGUGUGAUGUGGCCCAUG CAUGGGCCACAUCACACAG 2241 2242 1527-1545 UGUGUGAUGUGGCCCAUGA UCAUGGGCCACAUCACACA 2243 2244 1528-1546 GUGUGAUGUGGCCCAUGAG CUCAUGGGCCACAUCACAC 2245 2246 1529-1547 UGUGAUGUGGCCCAUGAGU ACUCAUGGGCCACAUCACA 2247 2248 1532-1550 GAUGUGGCCCAUGAGUUUG CAAACUCAUGGGCCACAUC 2249 2250 1533-1551 AUGUGGCCCAUGAGUUUGG CCAAACUCAUGGGCCACAU 2251 2252 1534-1552 UGUGGCCCAUGAGUUUGGA UCCAAACUCAUGGGCCACA 2253 2254 1535-1553 GUGGCCCAUGAGUUUGGAG CUCCAAACUCAUGGGCCAC 2255 2256 1536-1554 UGGCCCAUGAGUUUGGAGC GCUCCAAACUCAUGGGCCA 2257 2258 1537-1555 GGCCCAUGAGUUUGGAGCA UGCUCCAAACUCAUGGGCC 2259 2260 1538-1556 GCCCAUGAGUUUGGAGCAA UUGCUCCAAACUCAUGGGC 2261 2262 1539-1557 CCCAUGAGUUUGGAGCAAU AUUGCUCCAAACUCAUGGG 2263 2264 1540-1558 CCAUGAGUUUGGAGCAAUC GAUUGCUCCAAACUCAUGG 2265 2266 1542-1560 AUGAGUUUGGAGCAAUCAC GUGAUUGCUCCAAACUCAU 2267 2268 1543-1561 UGAGUUUGGAGCAAUCACC GGUGAUUGCUCCAAACUCA 2269 2270 1545-1563 AGUUUGGAGCAAUCACCUU AAGGUGAUUGCUCCAAACU 2271 2272 1546-1564 GUUUGGAGCAAUCACCUUC GAAGGUGAUUGCUCCAAAC 2273 2274 1547-1565 UUUGGAGCAAUCACCUUCG CGAAGGUGAUUGCUCCAAA 2275 2276 1548-1566 UUGGAGCAAUCACCUUCGU ACGAAGGUGAUUGCUCCAA 2277 2278 1549-1567 UGGAGCAAUCACCUUCGUG CACGAAGGUGAUUGCUCCA 2279 2280 1550-1568 GGAGCAAUCACCUUCGUGG CCACGAAGGUGAUUGCUCC 2281 2282 1551-1569 GAGCAAUCACCUUCGUGGA UCCACGAAGGUGAUUGCUC 2283 2284 1552-1570 AGCAAUCACCUUCGUGGAU AUCCACGAAGGUGAUUGCU 2285 2286 1553-1571 GCAAUCACCUUCGUGGAUG CAUCCACGAAGGUGAUUGC 2287 2288 1554-1572 CAAUCACCUUCGUGGAUGA UCAUCCACGAAGGUGAUUG 2289 2290 1555-1573 AAUCACCUUCGUGGAUGAG CUCAUCCACGAAGGUGAUU 2291 2292 1556-1574 AUCACCUUCGUGGAUGAGG CCUCAUCCACGAAGGUGAU 2293 2294 1557-1575 UCACCUUCGUGGAUGAGGU ACCUCAUCCACGAAGGUGA 2295 2296 1558-1576 CACCUUCGUGGAUGAGGUC GACCUCAUCCACGAAGGUG 2297 2298 1559-1577 ACCUUCGUGGAUGAGGUCC GGACCUCAUCCACGAAGGU 2299 2300 1560-1578 CCUUCGUGGAUGAGGUCCA UGGACCUCAUCCACGAAGG 2301 2302 1561-1579 CUUCGUGGAUGAGGUCCAC GUGGACCUCAUCCACGAAG 2303 2304 1562-1580 UUCGUGGAUGAGGUCCACG CGUGGACCUCAUCCACGAA 2305 2306 1563-1581 UCGUGGAUGAGGUCCACGC GCGUGGACCUCAUCCACGA 2307 2308 1564-1582 CGUGGAUGAGGUCCACGCA UGCGUGGACCUCAUCCACG 2309 2310 1565-1583 GUGGAUGAGGUCCACGCAG CUGCGUGGACCUCAUCCAC 2311 2312 1566-1584 UGGAUGAGGUCCACGCAGU ACUGCGUGGACCUCAUCCA 2313 2314 1567-1585 GGAUGAGGUCCACGCAGUG CACUGCGUGGACCUCAUCC 2315 2316 1568-1586 GAUGAGGUCCACGCAGUGG CCACUGCGUGGACCUCAUC 2317 2318 1569-1587 AUGAGGUCCACGCAGUGGG CCCACUGCGUGGACCUCAU 2319 2320 1570-1588 UGAGGUCCACGCAGUGGGG CCCCACUGCGUGGACCUCA 2321 2322 1571-1589 GAGGUCCACGCAGUGGGGC GCCCCACUGCGUGGACCUC 2323 2324 1572-1590 AGGUCCACGCAGUGGGGCU AGCCCCACUGCGUGGACCU 2325 2326 1595-1613 GGGGCUCGAGGCGGAGGGA UCCCUCCGCCUCGAGCCCC 2327 2328 1596-1614 GGGCUCGAGGCGGAGGGAU AUCCCUCCGCCUCGAGCCC 2329 2330 1597-1615 GGCUCGAGGCGGAGGGAUU AAUCCCUCCGCCUCGAGCC 2331 2332 1598-1616 GCUCGAGGCGGAGGGAUUG CAAUCCCUCCGCCUCGAGC 2333 2334 1599-1617 CUCGAGGCGGAGGGAUUGG CCAAUCCCUCCGCCUCGAG 2335 2336 1600-1618 UCGAGGCGGAGGGAUUGGG CCCAAUCCCUCCGCCUCGA 2337 2338 1601-1619 CGAGGCGGAGGGAUUGGGG CCCCAAUCCCUCCGCCUCG 2339 2340 1602-1620 GAGGCGGAGGGAUUGGGGA UCCCCAAUCCCUCCGCCUC 2341 2342 1603-1621 AGGCGGAGGGAUUGGGGAU AUCCCCAAUCCCUCCGCCU 2343 2344 1604-1622 GGCGGAGGGAUUGGGGAUC GAUCCCCAAUCCCUCCGCC 2345 2346 1605-1623 GCGGAGGGAUUGGGGAUCG CGAUCCCCAAUCCCUCCGC 2347 2348 1606-1624 CGGAGGGAUUGGGGAUCGG CCGAUCCCCAAUCCCUCCG 2349 2350 1607-1625 GGAGGGAUUGGGGAUCGGG CCCGAUCCCCAAUCCCUCC 2351 2352 1608-1626 GAGGGAUUGGGGAUCGGGA UCCCGAUCCCCAAUCCCUC 2353 2354 1609-1627 AGGGAUUGGGGAUCGGGAU AUCCCGAUCCCCAAUCCCU 2355 2356 1610-1628 GGGAUUGGGGAUCGGGAUG CAUCCCGAUCCCCAAUCCC 2357 2358 1611-1629 GGAUUGGGGAUCGGGAUGG CCAUCCCGAUCCCCAAUCC 2359 2360 1612-1630 GAUUGGGGAUCGGGAUGGA UCCAUCCCGAUCCCCAAUC 2361 2362 1613-1631 AUUGGGGAUCGGGAUGGAG CUCCAUCCCGAUCCCCAAU 2363 2364 1614-1632 UUGGGGAUCGGGAUGGAGU ACUCCAUCCCGAUCCCCAA 2365 2366 1615-1633 UGGGGAUCGGGAUGGAGUC GACUCCAUCCCGAUCCCCA 2367 2368 1617-1635 GGGAUCGGGAUGGAGUCAU AUGACUCCAUCCCGAUCCC 2369 2370 1618-1636 GGAUCGGGAUGGAGUCAUG CAUGACUCCAUCCCGAUCC 2371 2372 1619-1637 GAUCGGGAUGGAGUCAUGC GCAUGACUCCAUCCCGAUC 2373 2374 1620-1638 AUCGGGAUGGAGUCAUGCC GGCAUGACUCCAUCCCGAU 2375 2376 1621-1639 UCGGGAUGGAGUCAUGCCA UGGCAUGACUCCAUCCCGA 2377 2378 1622-1640 CGGGAUGGAGUCAUGCCAA UUGGCAUGACUCCAUCCCG 2379 2380 1623-1641 GGGAUGGAGUCAUGCCAAA UUUGGCAUGACUCCAUCCC 2381 2382 1624-1642 GGAUGGAGUCAUGCCAAAA UUUUGGCAUGACUCCAUCC 2383 2384 1625-1643 GAUGGAGUCAUGCCAAAAA UUUUUGGCAUGACUCCAUC 2385 2386 1626-1644 AUGGAGUCAUGCCAAAAAU AUUUUUGGCAUGACUCCAU 2387 2388 1627-1645 UGGAGUCAUGCCAAAAAUG CAUUUUUGGCAUGACUCCA 2389 2390 1628-1646 GGAGUCAUGCCAAAAAUGG CCAUUUUUGGCAUGACUCC 2391 2392 1629-1647 GAGUCAUGCCAAAAAUGGA UCCAUUUUUGGCAUGACUC 2393 2394 1630-1648 AGUCAUGCCAAAAAUGGAC GUCCAUUUUUGGCAUGACU 2395 2396 1632-1650 UCAUGCCAAAAAUGGACAU AUGUCCAUUUUUGGCAUGA 2397 2398 1633-1651 CAUGCCAAAAAUGGACAUC GAUGUCCAUUUUUGGCAUG 2399 2400 1636-1654 GCCAAAAAUGGACAUCAUU AAUGAUGUCCAUUUUUGGC 2401 2402 1638-1656 CAAAAAUGGACAUCAUUUC GAAAUGAUGUCCAUUUUUG 2403 2404 1639-1657 AAAAAUGGACAUCAUUUCU AGAAAUGAUGUCCAUUUUU 2405 2406 1640-1658 AAAAUGGACAUCAUUUCUG CAGAAAUGAUGUCCAUUUU 2407 2408 1641-1659 AAAUGGACAUCAUUUCUGG CCAGAAAUGAUGUCCAUUU 2409 2410 1642-1660 AAUGGACAUCAUUUCUGGA UCCAGAAAUGAUGUCCAUU 2411 2412 1643-1661 AUGGACAUCAUUUCUGGAA UUCCAGAAAUGAUGUCCAU 2413 2414 1644-1662 UGGACAUCAUUUCUGGAAC GUUCCAGAAAUGAUGUCCA 2415 2416 1645-1663 GGACAUCAUUUCUGGAACA UGUUCCAGAAAUGAUGUCC 2417 2418 1646-1664 GACAUCAUUUCUGGAACAC GUGUUCCAGAAAUGAUGUC 2419 2420 1647-1665 ACAUCAUUUCUGGAACACU AGUGUUCCAGAAAUGAUGU 2421 2422 1648-1666 CAUCAUUUCUGGAACACUU AAGUGUUCCAGAAAUGAUG 2423 2424 1649-1667 AUCAUUUCUGGAACACUUG CAAGUGUUCCAGAAAUGAU 2425 2426 1650-1668 UCAUUUCUGGAACACUUGG CCAAGUGUUCCAGAAAUGA 2427 2428 1651-1669 CAUUUCUGGAACACUUGGC GCCAAGUGUUCCAGAAAUG 2429 2430 1652-1670 AUUUCUGGAACACUUGGCA UGCCAAGUGUUCCAGAAAU 2431 2432 1653-1671 UUUCUGGAACACUUGGCAA UUGCCAAGUGUUCCAGAAA 2433 2434 1654-1672 UUCUGGAACACUUGGCAAA UUUGCCAAGUGUUCCAGAA 2435 2436 1655-1673 UCUGGAACACUUGGCAAAG CUUUGCCAAGUGUUCCAGA 2437 2438 1656-1674 CUGGAACACUUGGCAAAGC GCUUUGCCAAGUGUUCCAG 2439 2440 1657-1675 UGGAACACUUGGCAAAGCC GGCUUUGCCAAGUGUUCCA 2441 2442 1658-1676 GGAACACUUGGCAAAGCCU AGGCUUUGCCAAGUGUUCC 2443 2444 1659-1677 GAACACUUGGCAAAGCCUU AAGGCUUUGCCAAGUGUUC 2445 2446 1660-1678 AACACUUGGCAAAGCCUUU AAAGGCUUUGCCAAGUGUU 2447 2448 1661-1679 ACACUUGGCAAAGCCUUUG CAAAGGCUUUGCCAAGUGU 2449 2450 1662-1680 CACUUGGCAAAGCCUUUGG CCAAAGGCUUUGCCAAGUG 2451 2452 1682-1700 UGUGUUGGAGGGUACAUCG CGAUGUACCCUCCAACACA 2453 2454 1683-1701 GUGUUGGAGGGUACAUCGC GCGAUGUACCCUCCAACAC 2455 2456 1684-1702 UGUUGGAGGGUACAUCGCC GGCGAUGUACCCUCCAACA 2457 2458 1685-1703 GUUGGAGGGUACAUCGCCA UGGCGAUGUACCCUCCAAC 2459 2460 1686-1704 UUGGAGGGUACAUCGCCAG CUGGCGAUGUACCCUCCAA 2461 2462 1687-1705 UGGAGGGUACAUCGCCAGC GCUGGCGAUGUACCCUCCA 2463 2464 1688-1706 GGAGGGUACAUCGCCAGCA UGCUGGCGAUGUACCCUCC 2465 2466 1689-1707 GAGGGUACAUCGCCAGCAC GUGCUGGCGAUGUACCCUC 2467 2468 1690-1708 AGGGUACAUCGCCAGCACG CGUGCUGGCGAUGUACCCU 2469 2470 1691-1709 GGGUACAUCGCCAGCACGA UCGUGCUGGCGAUGUACCC 2471 2472 1692-1710 GGUACAUCGCCAGCACGAG CUCGUGCUGGCGAUGUACC 2473 2474 1693-1711 GUACAUCGCCAGCACGAGU ACUCGUGCUGGCGAUGUAC 2475 2476 1694-1712 UACAUCGCCAGCACGAGUU AACUCGUGCUGGCGAUGUA 2477 2478 1695-1713 ACAUCGCCAGCACGAGUUC GAACUCGUGCUGGCGAUGU 2479 2480 1696-1714 CAUCGCCAGCACGAGUUCU AGAACUCGUGCUGGCGAUG 2481 2482 1697-1715 AUCGCCAGCACGAGUUCUC GAGAACUCGUGCUGGCGAU 2483 2484 1698-1716 UCGCCAGCACGAGUUCUCU AGAGAACUCGUGCUGGCGA 2485 2486 1699-1717 CGCCAGCACGAGUUCUCUG CAGAGAACUCGUGCUGGCG 2487 2488 1700-1718 GCCAGCACGAGUUCUCUGA UCAGAGAACUCGUGCUGGC 2489 2490 1701-1719 CCAGCACGAGUUCUCUGAU AUCAGAGAACUCGUGCUGG 2491 2492 1702-1720 CAGCACGAGUUCUCUGAUU AAUCAGAGAACUCGUGCUG 2493 2494 1703-1721 AGCACGAGUUCUCUGAUUG CAAUCAGAGAACUCGUGCU 2495 2496 1704-1722 GCACGAGUUCUCUGAUUGA UCAAUCAGAGAACUCGUGC 2497 2498 1705-1723 CACGAGUUCUCUGAUUGAC GUCAAUCAGAGAACUCGUG 2499 2500 1707-1725 CGAGUUCUCUGAUUGACAC GUGUCAAUCAGAGAACUCG 2501 2502 1727-1745 GUACGGUCCUAUGCUGCUG CAGCAGCAUAGGACCGUAC 2503 2504 1728-1746 UACGGUCCUAUGCUGCUGG CCAGCAGCAUAGGACCGUA 2505 2506 1729-1747 ACGGUCCUAUGCUGCUGGC GCCAGCAGCAUAGGACCGU 2507 2508 1730-1748 CGGUCCUAUGCUGCUGGCU AGCCAGCAGCAUAGGACCG 2509 2510 1731-1749 GGUCCUAUGCUGCUGGCUU AAGCCAGCAGCAUAGGACC 2511 2512 1732-1750 GUCCUAUGCUGCUGGCUUC GAAGCCAGCAGCAUAGGAC 2513 2514 1733-1751 UCCUAUGCUGCUGGCUUCA UGAAGCCAGCAGCAUAGGA 2515 2516 1734-1752 CCUAUGCUGCUGGCUUCAU AUGAAGCCAGCAGCAUAGG 2517 2518 1735-1753 CUAUGCUGCUGGCUUCAUC GAUGAAGCCAGCAGCAUAG 2519 2520 1736-1754 UAUGCUGCUGGCUUCAUCU AGAUGAAGCCAGCAGCAUA 2521 2522 1737-1755 AUGCUGCUGGCUUCAUCUU AAGAUGAAGCCAGCAGCAU 2523 2524 1738-1756 UGCUGCUGGCUUCAUCUUC GAAGAUGAAGCCAGCAGCA 2525 2526 1739-1757 GCUGCUGGCUUCAUCUUCA UGAAGAUGAAGCCAGCAGC 2527 2528 1740-1758 CUGCUGGCUUCAUCUUCAC GUGAAGAUGAAGCCAGCAG 2529 2530 1741-1759 UGCUGGCUUCAUCUUCACC GGUGAAGAUGAAGCCAGCA 2531 2532 1742-1760 GCUGGCUUCAUCUUCACCA UGGUGAAGAUGAAGCCAGC 2533 2534 1743-1761 CUGGCUUCAUCUUCACCAC GUGGUGAAGAUGAAGCCAG 2535 2536 1744-1762 UGGCUUCAUCUUCACCACC GGUGGUGAAGAUGAAGCCA 2537 2538 1745-1763 GGCUUCAUCUUCACCACCU AGGUGGUGAAGAUGAAGCC 2539 2540 1746-1764 GCUUCAUCUUCACCACCUC GAGGUGGUGAAGAUGAAGC 2541 2542 1747-1765 CUUCAUCUUCACCACCUCU AGAGGUGGUGAAGAUGAAG 2543 2544 1748-1766 UUCAUCUUCACCACCUCUC GAGAGGUGGUGAAGAUGAA 2545 2546 1749-1767 UCAUCUUCACCACCUCUCU AGAGAGGUGGUGAAGAUGA 2547 2548 1750-1768 CAUCUUCACCACCUCUCUG CAGAGAGGUGGUGAAGAUG 2549 2550 1751-1769 AUCUUCACCACCUCUCUGC GCAGAGAGGUGGUGAAGAU 2551 2552 1752-1770 UCUUCACCACCUCUCUGCC GGCAGAGAGGUGGUGAAGA 2553 2554 1753-1771 CUUCACCACCUCUCUGCCA UGGCAGAGAGGUGGUGAAG 2555 2556 1754-1772 UUCACCACCUCUCUGCCAC GUGGCAGAGAGGUGGUGAA 2557 2558 1755-1773 UCACCACCUCUCUGCCACC GGUGGCAGAGAGGUGGUGA 2559 2560 1756-1774 CACCACCUCUCUGCCACCC GGGUGGCAGAGAGGUGGUG 2561 2562 1757-1775 ACCACCUCUCUGCCACCCA UGGGUGGCAGAGAGGUGGU 2563 2564 1758-1776 CCACCUCUCUGCCACCCAU AUGGGUGGCAGAGAGGUGG 2565 2566 1759-1777 CACCUCUCUGCCACCCAUG CAUGGGUGGCAGAGAGGUG 2567 2568 1760-1778 ACCUCUCUGCCACCCAUGC GCAUGGGUGGCAGAGAGGU 2569 2570 1761-1779 CCUCUCUGCCACCCAUGCU AGCAUGGGUGGCAGAGAGG 2571 2572 1762-1780 CUCUCUGCCACCCAUGCUG CAGCAUGGGUGGCAGAGAG 2573 2574 1763-1781 UCUCUGCCACCCAUGCUGC GCAGCAUGGGUGGCAGAGA 2575 2576 1764-1782 CUCUGCCACCCAUGCUGCU AGCAGCAUGGGUGGCAGAG 2577 2578 1765-1783 UCUGCCACCCAUGCUGCUG CAGCAGCAUGGGUGGCAGA 2579 2580 1766-1784 CUGCCACCCAUGCUGCUGG CCAGCAGCAUGGGUGGCAG 2581 2582 1767-1785 UGCCACCCAUGCUGCUGGC GCCAGCAGCAUGGGUGGCA 2583 2584 1768-1786 GCCACCCAUGCUGCUGGCU AGCCAGCAGCAUGGGUGGC 2585 2586 1769-1787 CCACCCAUGCUGCUGGCUG CAGCCAGCAGCAUGGGUGG 2587 2588 1770-1788 CACCCAUGCUGCUGGCUGG CCAGCCAGCAGCAUGGGUG 2589 2590 1771-1789 ACCCAUGCUGCUGGCUGGA UCCAGCCAGCAGCAUGGGU 2591 2592 1772-1790 CCCAUGCUGCUGGCUGGAG CUCCAGCCAGCAGCAUGGG 2593 2594 1773-1791 CCAUGCUGCUGGCUGGAGC GCUCCAGCCAGCAGCAUGG 2595 2596 1774-1792 CAUGCUGCUGGCUGGAGCC GGCUCCAGCCAGCAGCAUG 2597 2598 1775-1793 AUGCUGCUGGCUGGAGCCC GGGCUCCAGCCAGCAGCAU 2599 2600 1776-1794 UGCUGCUGGCUGGAGCCCU AGGGCUCCAGCCAGCAGCA 2601 2602 1777-1795 GCUGCUGGCUGGAGCCCUG CAGGGCUCCAGCCAGCAGC 2603 2604 1778-1796 CUGCUGGCUGGAGCCCUGG CCAGGGCUCCAGCCAGCAG 2605 2606 1779-1797 UGCUGGCUGGAGCCCUGGA UCCAGGGCUCCAGCCAGCA 2607 2608 1780-1798 GCUGGCUGGAGCCCUGGAG CUCCAGGGCUCCAGCCAGC 2609 2610 1781-1799 CUGGCUGGAGCCCUGGAGU ACUCCAGGGCUCCAGCCAG 2611 2612 1782-1800 UGGCUGGAGCCCUGGAGUC GACUCCAGGGCUCCAGCCA 2613 2614 1783-1801 GGCUGGAGCCCUGGAGUCU AGACUCCAGGGCUCCAGCC 2615 2616 1784-1802 GCUGGAGCCCUGGAGUCUG CAGACUCCAGGGCUCCAGC 2617 2618 1785-1803 CUGGAGCCCUGGAGUCUGU ACAGACUCCAGGGCUCCAG 2619 2620 1786-1804 UGGAGCCCUGGAGUCUGUG CACAGACUCCAGGGCUCCA 2621 2622 1787-1805 GGAGCCCUGGAGUCUGUGC GCACAGACUCCAGGGCUCC 2623 2624 1788-1806 GAGCCCUGGAGUCUGUGCG CGCACAGACUCCAGGGCUC 2625 2626 1789-1807 AGCCCUGGAGUCUGUGCGG CCGCACAGACUCCAGGGCU 2627 2628 1790-1808 GCCCUGGAGUCUGUGCGGA UCCGCACAGACUCCAGGGC 2629 2630 1792-1810 CCUGGAGUCUGUGCGGAUC GAUCCGCACAGACUCCAGG 2631 2632 1793-1811 CUGGAGUCUGUGCGGAUCC GGAUCCGCACAGACUCCAG 2633 2634 1795-1813 GGAGUCUGUGCGGAUCCUG CAGGAUCCGCACAGACUCC 2635 2636 1796-1814 GAGUCUGUGCGGAUCCUGA UCAGGAUCCGCACAGACUC 2637 2638 1797-1815 AGUCUGUGCGGAUCCUGAA UUCAGGAUCCGCACAGACU 2639 2640 1798-1816 GUCUGUGCGGAUCCUGAAG CUUCAGGAUCCGCACAGAC 2641 2642 1799-1817 UCUGUGCGGAUCCUGAAGA UCUUCAGGAUCCGCACAGA 2643 2644 1800-1818 CUGUGCGGAUCCUGAAGAG CUCUUCAGGAUCCGCACAG 2645 2646 1801-1819 UGUGCGGAUCCUGAAGAGC GCUCUUCAGGAUCCGCACA 2647 2648 1802-1820 GUGCGGAUCCUGAAGAGCG CGCUCUUCAGGAUCCGCAC 2649 2650 1803-1821 UGCGGAUCCUGAAGAGCGC GCGCUCUUCAGGAUCCGCA 2651 2652 1804-1822 GCGGAUCCUGAAGAGCGCU AGCGCUCUUCAGGAUCCGC 2653 2654 1805-1823 CGGAUCCUGAAGAGCGCUG CAGCGCUCUUCAGGAUCCG 2655 2656 1806-1824 GGAUCCUGAAGAGCGCUGA UCAGCGCUCUUCAGGAUCC 2657 2658 1807-1825 GAUCCUGAAGAGCGCUGAG CUCAGCGCUCUUCAGGAUC 2659 2660 1808-1826 AUCCUGAAGAGCGCUGAGG CCUCAGCGCUCUUCAGGAU 2661 2662 1809-1827 UCCUGAAGAGCGCUGAGGG CCCUCAGCGCUCUUCAGGA 2663 2664 1810-1828 CCUGAAGAGCGCUGAGGGA UCCCUCAGCGCUCUUCAGG 2665 2666 1811-1829 CUGAAGAGCGCUGAGGGAC GUCCCUCAGCGCUCUUCAG 2667 2668 1812-1830 UGAAGAGCGCUGAGGGACG CGUCCCUCAGCGCUCUUCA 2669 2670 1813-1831 GAAGAGCGCUGAGGGACGG CCGUCCCUCAGCGCUCUUC 2671 2672 1814-1832 AAGAGCGCUGAGGGACGGG CCCGUCCCUCAGCGCUCUU 2673 2674 1815-1833 AGAGCGCUGAGGGACGGGU ACCCGUCCCUCAGCGCUCU 2675 2676 1816-1834 GAGCGCUGAGGGACGGGUG CACCCGUCCCUCAGCGCUC 2677 2678 1817-1835 AGCGCUGAGGGACGGGUGC GCACCCGUCCCUCAGCGCU 2679 2680 1818-1836 GCGCUGAGGGACGGGUGCU AGCACCCGUCCCUCAGCGC 2681 2682 1819-1837 CGCUGAGGGACGGGUGCUU AAGCACCCGUCCCUCAGCG 2683 2684 1820-1838 GCUGAGGGACGGGUGCUUC GAAGCACCCGUCCCUCAGC 2685 2686 1821-1839 CUGAGGGACGGGUGCUUCG CGAAGCACCCGUCCCUCAG 2687 2688 1822-1840 UGAGGGACGGGUGCUUCGC GCGAAGCACCCGUCCCUCA 2689 2690 1823-1841 GAGGGACGGGUGCUUCGCC GGCGAAGCACCCGUCCCUC 2691 2692 1824-1842 AGGGACGGGUGCUUCGCCG CGGCGAAGCACCCGUCCCU 2693 2694 1825-1843 GGGACGGGUGCUUCGCCGC GCGGCGAAGCACCCGUCCC 2695 2696 1826-1844 GGACGGGUGCUUCGCCGCC GGCGGCGAAGCACCCGUCC 2697 2698 1827-1845 GACGGGUGCUUCGCCGCCA UGGCGGCGAAGCACCCGUC 2699 2700 1828-1846 ACGGGUGCUUCGCCGCCAG CUGGCGGCGAAGCACCCGU 2701 2702 1829-1847 CGGGUGCUUCGCCGCCAGC GCUGGCGGCGAAGCACCCG 2703 2704 1830-1848 GGGUGCUUCGCCGCCAGCA UGCUGGCGGCGAAGCACCC 2705 2706 1831-1849 GGUGCUUCGCCGCCAGCAC GUGCUGGCGGCGAAGCACC 2707 2708 1832-1850 GUGCUUCGCCGCCAGCACC GGUGCUGGCGGCGAAGCAC 2709 2710 1833-1851 UGCUUCGCCGCCAGCACCA UGGUGCUGGCGGCGAAGCA 2711 2712 1834-1852 GCUUCGCCGCCAGCACCAG CUGGUGCUGGCGGCGAAGC 2713 2714 1835-1853 CUUCGCCGCCAGCACCAGC GCUGGUGCUGGCGGCGAAG 2715 2716 1836-1854 UUCGCCGCCAGCACCAGCG CGCUGGUGCUGGCGGCGAA 2717 2718 1837-1855 UCGCCGCCAGCACCAGCGC GCGCUGGUGCUGGCGGCGA 2719 2720 1838-1856 CGCCGCCAGCACCAGCGCA UGCGCUGGUGCUGGCGGCG 2721 2722 1839-1857 GCCGCCAGCACCAGCGCAA UUGCGCUGGUGCUGGCGGC 2723 2724 1840-1858 CCGCCAGCACCAGCGCAAC GUUGCGCUGGUGCUGGCGG 2725 2726 1841-1859 CGCCAGCACCAGCGCAACG CGUUGCGCUGGUGCUGGCG 2727 2728 1842-1860 GCCAGCACCAGCGCAACGU ACGUUGCGCUGGUGCUGGC 2729 2730 1865-1883 CUCAUGAGACAGAUGCUAA UUAGCAUCUGUCUCAUGAG 2731 2732 1866-1884 UCAUGAGACAGAUGCUAAU AUUAGCAUCUGUCUCAUGA 2733 2734 1867-1885 CAUGAGACAGAUGCUAAUG CAUUAGCAUCUGUCUCAUG 2735 2736 1868-1886 AUGAGACAGAUGCUAAUGG CCAUUAGCAUCUGUCUCAU 2737 2738 1869-1887 UGAGACAGAUGCUAAUGGA UCCAUUAGCAUCUGUCUCA 2739 2740 1871-1889 AGACAGAUGCUAAUGGAUG CAUCCAUUAGCAUCUGUCU 2741 2742 1872-1890 GACAGAUGCUAAUGGAUGC GCAUCCAUUAGCAUCUGUC 2743 2744 1873-1891 ACAGAUGCUAAUGGAUGCC GGCAUCCAUUAGCAUCUGU 2745 2746 1874-1892 CAGAUGCUAAUGGAUGCCG CGGCAUCCAUUAGCAUCUG 2747 2748 1875-1893 AGAUGCUAAUGGAUGCCGG CCGGCAUCCAUUAGCAUCU 2749 2750 1876-1894 GAUGCUAAUGGAUGCCGGC GCCGGCAUCCAUUAGCAUC 2751 2752 1877-1895 AUGCUAAUGGAUGCCGGCC GGCCGGCAUCCAUUAGCAU 2753 2754 1878-1896 UGCUAAUGGAUGCCGGCCU AGGCCGGCAUCCAUUAGCA 2755 2756 1879-1897 GCUAAUGGAUGCCGGCCUC GAGGCCGGCAUCCAUUAGC 2757 2758 1880-1898 CUAAUGGAUGCCGGCCUCC GGAGGCCGGCAUCCAUUAG 2759 2760 1881-1899 UAAUGGAUGCCGGCCUCCC GGGAGGCCGGCAUCCAUUA 2761 2762 1882-1900 AAUGGAUGCCGGCCUCCCU AGGGAGGCCGGCAUCCAUU 2763 2764 1883-1901 AUGGAUGCCGGCCUCCCUG CAGGGAGGCCGGCAUCCAU 2765 2766 1884-1902 UGGAUGCCGGCCUCCCUGU ACAGGGAGGCCGGCAUCCA 2767 2768 1885-1903 GGAUGCCGGCCUCCCUGUU AACAGGGAGGCCGGCAUCC 2769 2770 1886-1904 GAUGCCGGCCUCCCUGUUG CAACAGGGAGGCCGGCAUC 2771 2772 1887-1905 AUGCCGGCCUCCCUGUUGU ACAACAGGGAGGCCGGCAU 2773 2774 1888-1906 UGCCGGCCUCCCUGUUGUC GACAACAGGGAGGCCGGCA 2775 2776 1889-1907 GCCGGCCUCCCUGUUGUCC GGACAACAGGGAGGCCGGC 2777 2778 1890-1908 CCGGCCUCCCUGUUGUCCA UGGACAACAGGGAGGCCGG 2779 2780 1891-1909 CGGCCUCCCUGUUGUCCAC GUGGACAACAGGGAGGCCG 2781 2782 1892-1910 GGCCUCCCUGUUGUCCACU AGUGGACAACAGGGAGGCC 2783 2784 1893-1911 GCCUCCCUGUUGUCCACUG CAGUGGACAACAGGGAGGC 2785 2786 1894-1912 CCUCCCUGUUGUCCACUGC GCAGUGGACAACAGGGAGG 2787 2788 1895-1913 CUCCCUGUUGUCCACUGCC GGCAGUGGACAACAGGGAG 2789 2790 1896-1914 UCCCUGUUGUCCACUGCCC GGGCAGUGGACAACAGGGA 2791 2792 1897-1915 CCCUGUUGUCCACUGCCCC GGGGCAGUGGACAACAGGG 2793 2794 1898-1916 CCUGUUGUCCACUGCCCCA UGGGGCAGUGGACAACAGG 2795 2796 1899-1917 CUGUUGUCCACUGCCCCAG CUGGGGCAGUGGACAACAG 2797 2798 1900-1918 UGUUGUCCACUGCCCCAGC GCUGGGGCAGUGGACAACA 2799 2800 1901-1919 GUUGUCCACUGCCCCAGCC GGCUGGGGCAGUGGACAAC 2801 2802 1902-1920 UUGUCCACUGCCCCAGCCA UGGCUGGGGCAGUGGACAA 2803 2804 1903-1921 UGUCCACUGCCCCAGCCAC GUGGCUGGGGCAGUGGACA 2805 2806 1904-1922 GUCCACUGCCCCAGCCACA UGUGGCUGGGGCAGUGGAC 2807 2808 1905-1923 UCCACUGCCCCAGCCACAU AUGUGGCUGGGGCAGUGGA 2809 2810 1906-1924 CCACUGCCCCAGCCACAUC GAUGUGGCUGGGGCAGUGG 2811 2812 1907-1925 CACUGCCCCAGCCACAUCA UGAUGUGGCUGGGGCAGUG 2813 2814 1908-1926 ACUGCCCCAGCCACAUCAU AUGAUGUGGCUGGGGCAGU 2815 2816 1909-1927 CUGCCCCAGCCACAUCAUC GAUGAUGUGGCUGGGGCAG 2817 2818 1910-1928 UGCCCCAGCCACAUCAUCC GGAUGAUGUGGCUGGGGCA 2819 2820 1911-1929 GCCCCAGCCACAUCAUCCC GGGAUGAUGUGGCUGGGGC 2821 2822 1912-1930 CCCCAGCCACAUCAUCCCU AGGGAUGAUGUGGCUGGGG 2823 2824 1913-1931 CCCAGCCACAUCAUCCCUG CAGGGAUGAUGUGGCUGGG 2825 2826 1914-1932 CCAGCCACAUCAUCCCUGU ACAGGGAUGAUGUGGCUGG 2827 2828 1915-1933 CAGCCACAUCAUCCCUGUG CACAGGGAUGAUGUGGCUG 2829 2830 1916-1934 AGCCACAUCAUCCCUGUGC GCACAGGGAUGAUGUGGCU 2831 2832 1917-1935 GCCACAUCAUCCCUGUGCG CGCACAGGGAUGAUGUGGC 2833 2834 1918-1936 CCACAUCAUCCCUGUGCGG CCGCACAGGGAUGAUGUGG 2835 2836 1919-1937 CACAUCAUCCCUGUGCGGG CCCGCACAGGGAUGAUGUG 2837 2838 1920-1938 ACAUCAUCCCUGUGCGGGU ACCCGCACAGGGAUGAUGU 2839 2840 1922-1940 AUCAUCCCUGUGCGGGUUG CAACCCGCACAGGGAUGAU 2841 2842 1923-1941 UCAUCCCUGUGCGGGUUGC GCAACCCGCACAGGGAUGA 2843 2844 1924-1942 CAUCCCUGUGCGGGUUGCA UGCAACCCGCACAGGGAUG 2845 2846 1925-1943 AUCCCUGUGCGGGUUGCAG CUGCAACCCGCACAGGGAU 2847 2848 1926-1944 UCCCUGUGCGGGUUGCAGA UCUGCAACCCGCACAGGGA 2849 2850 1928-1946 CCUGUGCGGGUUGCAGAUG CAUCUGCAACCCGCACAGG 2851 2852 1929-1947 CUGUGCGGGUUGCAGAUGC GCAUCUGCAACCCGCACAG 2853 2854 1930-1948 UGUGCGGGUUGCAGAUGCU AGCAUCUGCAACCCGCACA 2855 2856 1931-1949 GUGCGGGUUGCAGAUGCUG CAGCAUCUGCAACCCGCAC 2857 2858 1932-1950 UGCGGGUUGCAGAUGCUGC GCAGCAUCUGCAACCCGCA 2859 2860 1933-1951 GCGGGUUGCAGAUGCUGCU AGCAGCAUCUGCAACCCGC 2861 2862 1934-1952 CGGGUUGCAGAUGCUGCUA UAGCAGCAUCUGCAACCCG 2863 2864 1935-1953 GGGUUGCAGAUGCUGCUAA UUAGCAGCAUCUGCAACCC 2865 2866 1936-1954 GGUUGCAGAUGCUGCUAAA UUUAGCAGCAUCUGCAACC 2867 2868 1937-1955 GUUGCAGAUGCUGCUAAAA UUUUAGCAGCAUCUGCAAC 2869 2870 1938-1956 UUGCAGAUGCUGCUAAAAA UUUUUAGCAGCAUCUGCAA 2871 2872 1939-1957 UGCAGAUGCUGCUAAAAAC GUUUUUAGCAGCAUCUGCA 2873 2874 1940-1958 GCAGAUGCUGCUAAAAACA UGUUUUUAGCAGCAUCUGC 2875 2876 1941-1959 CAGAUGCUGCUAAAAACAC GUGUUUUUAGCAGCAUCUG 2877 2878 1961-1979 GAAGUCUGUGAUGAACUAA UUAGUUCAUCACAGACUUC 2879 2880 1963-1981 AGUCUGUGAUGAACUAAUG CAUUAGUUCAUCACAGACU 2881 2882 1965-1983 UCUGUGAUGAACUAAUGAG CUCAUUAGUUCAUCACAGA 2883 2884 1966-1984 CUGUGAUGAACUAAUGAGC GCUCAUUAGUUCAUCACAG 2885 2886 1968-1986 GUGAUGAACUAAUGAGCAG CUGCUCAUUAGUUCAUCAC 2887 2888 1969-1987 UGAUGAACUAAUGAGCAGA UCUGCUCAUUAGUUCAUCA 2889 2890 1970-1988 GAUGAACUAAUGAGCAGAC GUCUGCUCAUUAGUUCAUC 2891 2892 1971-1989 AUGAACUAAUGAGCAGACA UGUCUGCUCAUUAGUUCAU 2893 2894 1972-1990 UGAACUAAUGAGCAGACAU AUGUCUGCUCAUUAGUUCA 2895 2896 1973-1991 GAACUAAUGAGCAGACAUA UAUGUCUGCUCAUUAGUUC 2897 2898 1974-1992 AACUAAUGAGCAGACAUAA UUAUGUCUGCUCAUUAGUU 2899 2900 1975-1993 ACUAAUGAGCAGACAUAAC GUUAUGUCUGCUCAUUAGU 2901 2902 1978-1996 AAUGAGCAGACAUAACAUC GAUGUUAUGUCUGCUCAUU 2903 2904 1979-1997 AUGAGCAGACAUAACAUCU AGAUGUUAUGUCUGCUCAU 2905 2906 1980-1998 UGAGCAGACAUAACAUCUA UAGAUGUUAUGUCUGCUCA 2907 2908 2000-2018 GUGCAAGCAAUCAAUUACC GGUAAUUGAUUGCUUGCAC 2909 2910 2001-2019 UGCAAGCAAUCAAUUACCC GGGUAAUUGAUUGCUUGCA 2911 2912 2002-2020 GCAAGCAAUCAAUUACCCU AGGGUAAUUGAUUGCUUGC 2913 2914 2004-2022 AAGCAAUCAAUUACCCUAC GUAGGGUAAUUGAUUGCUU 2915 2916 2024-2042 GUGCCCCGGGGAGAAGAGC GCUCUUCUCCCCGGGGCAC 2917 2918 2025-2043 UGCCCCGGGGAGAAGAGCU AGCUCUUCUCCCCGGGGCA 2919 2920 2026-2044 GCCCCGGGGAGAAGAGCUC GAGCUCUUCUCCCCGGGGC 2921 2922 2027-2045 CCCCGGGGAGAAGAGCUCC GGAGCUCUUCUCCCCGGGG 2923 2924 2028-2046 CCCGGGGAGAAGAGCUCCU AGGAGCUCUUCUCCCCGGG 2925 2926 2029-2047 CCGGGGAGAAGAGCUCCUA UAGGAGCUCUUCUCCCCGG 2927 2928 2030-2048 CGGGGAGAAGAGCUCCUAC GUAGGAGCUCUUCUCCCCG 2929 2930 2031-2049 GGGGAGAAGAGCUCCUACG CGUAGGAGCUCUUCUCCCC 2931 2932 2032-2050 GGGAGAAGAGCUCCUACGG CCGUAGGAGCUCUUCUCCC 2933 2934 2033-2051 GGAGAAGAGCUCCUACGGA UCCGUAGGAGCUCUUCUCC 2935 2936 2034-2052 GAGAAGAGCUCCUACGGAU AUCCGUAGGAGCUCUUCUC 2937 2938 2060-2078 ACCCCUCACCACACACCCC GGGGUGUGUGGUGAGGGGU 2939 2940 2061-2079 CCCCUCACCACACACCCCA UGGGGUGUGUGGUGAGGGG 2941 2942 2062-2080 CCCUCACCACACACCCCAG CUGGGGUGUGUGGUGAGGG 2943 2944 2063-2081 CCUCACCACACACCCCAGA UCUGGGGUGUGUGGUGAGG 2945 2946 2064-2082 CUCACCACACACCCCAGAU AUCUGGGGUGUGUGGUGAG 2947 2948 2065-2083 UCACCACACACCCCAGAUG CAUCUGGGGUGUGUGGUGA 2949 2950 2066-2084 CACCACACACCCCAGAUGA UCAUCUGGGGUGUGUGGUG 2951 2952 2067-2085 ACCACACACCCCAGAUGAU AUCAUCUGGGGUGUGUGGU 2953 2954 2068-2086 CCACACACCCCAGAUGAUG CAUCAUCUGGGGUGUGUGG 2955 2956 2069-2087 CACACACCCCAGAUGAUGA UCAUCAUCUGGGGUGUGUG 2957 2958 2070-2088 ACACACCCCAGAUGAUGAA UUCAUCAUCUGGGGUGUGU 2959 2960 2071-2089 CACACCCCAGAUGAUGAAC GUUCAUCAUCUGGGGUGUG 2961 2962 2072-2090 ACACCCCAGAUGAUGAACU AGUUCAUCAUCUGGGGUGU 2963 2964 2073-2091 CACCCCAGAUGAUGAACUA UAGUUCAUCAUCUGGGGUG 2965 2966 2074-2092 ACCCCAGAUGAUGAACUAC GUAGUUCAUCAUCUGGGGU 2967 2968 2076-2094 CCCAGAUGAUGAACUACUU AAGUAGUUCAUCAUCUGGG 2969 2970 2077-2095 CCAGAUGAUGAACUACUUC GAAGUAGUUCAUCAUCUGG 2971 2972 2078-2096 CAGAUGAUGAACUACUUCC GGAAGUAGUUCAUCAUCUG 2973 2974 2079-2097 AGAUGAUGAACUACUUCCU AGGAAGUAGUUCAUCAUCU 2975 2976 2080-2098 GAUGAUGAACUACUUCCUU AAGGAAGUAGUUCAUCAUC 2977 2978 2081-2099 AUGAUGAACUACUUCCUUG CAAGGAAGUAGUUCAUCAU 2979 2980 2082-2100 UGAUGAACUACUUCCUUGA UCAAGGAAGUAGUUCAUCA 2981 2982 2083-2101 GAUGAACUACUUCCUUGAG CUCAAGGAAGUAGUUCAUC 2983 2984 2084-2102 AUGAACUACUUCCUUGAGA UCUCAAGGAAGUAGUUCAU 2985 2986 2085-2103 UGAACUACUUCCUUGAGAA UUCUCAAGGAAGUAGUUCA 2987 2988 2086-2104 GAACUACUUCCUUGAGAAU AUUCUCAAGGAAGUAGUUC 2989 2990 2087-2105 AACUACUUCCUUGAGAAUC GAUUCUCAAGGAAGUAGUU 2991 2992 2088-2106 ACUACUUCCUUGAGAAUCU AGAUUCUCAAGGAAGUAGU 2993 2994 2089-2107 CUACUUCCUUGAGAAUCUG CAGAUUCUCAAGGAAGUAG 2995 2996 2090-2108 UACUUCCUUGAGAAUCUGC GCAGAUUCUCAAGGAAGUA 2997 2998 2091-2109 ACUUCCUUGAGAAUCUGCU AGCAGAUUCUCAAGGAAGU 2999 3000 2117-2135 UGGAAGCAAGUGGGGCUGG CCAGCCCCACUUGCUUCCA 3001 3002 2118-2136 GGAAGCAAGUGGGGCUGGA UCCAGCCCCACUUGCUUCC 3003 3004 2119-2137 GAAGCAAGUGGGGCUGGAA UUCCAGCCCCACUUGCUUC 3005 3006 2120-2138 AAGCAAGUGGGGCUGGAAC GUUCCAGCCCCACUUGCUU 3007 3008 2121-2139 AGCAAGUGGGGCUGGAACU AGUUCCAGCCCCACUUGCU 3009 3010 2122-2140 GCAAGUGGGGCUGGAACUG CAGUUCCAGCCCCACUUGC 3011 3012 2123-2141 CAAGUGGGGCUGGAACUGA UCAGUUCCAGCCCCACUUG 3013 3014 2124-2142 AAGUGGGGCUGGAACUGAA UUCAGUUCCAGCCCCACUU 3015 3016 2125-2143 AGUGGGGCUGGAACUGAAG CUUCAGUUCCAGCCCCACU 3017 3018 2126-2144 GUGGGGCUGGAACUGAAGC GCUUCAGUUCCAGCCCCAC 3019 3020 2127-2145 UGGGGCUGGAACUGAAGCC GGCUUCAGUUCCAGCCCCA 3021 3022 2147-2165 CAUUCCUCAGCUGAGUGCA UGCACUCAGCUGAGGAAUG 3023 3024 2148-2166 AUUCCUCAGCUGAGUGCAA UUGCACUCAGCUGAGGAAU 3025 3026 2149-2167 UUCCUCAGCUGAGUGCAAC GUUGCACUCAGCUGAGGAA 3027 3028 2150-2168 UCCUCAGCUGAGUGCAACU AGUUGCACUCAGCUGAGGA 3029 3030 2151-2169 CCUCAGCUGAGUGCAACUU AAGUUGCACUCAGCUGAGG 3031 3032 2152-2170 CUCAGCUGAGUGCAACUUC GAAGUUGCACUCAGCUGAG 3033 3034 2153-2171 UCAGCUGAGUGCAACUUCU AGAAGUUGCACUCAGCUGA 3035 3036 2154-2172 CAGCUGAGUGCAACUUCUG CAGAAGUUGCACUCAGCUG 3037 3038 2155-2173 AGCUGAGUGCAACUUCUGC GCAGAAGUUGCACUCAGCU 3039 3040 2156-2174 GCUGAGUGCAACUUCUGCA UGCAGAAGUUGCACUCAGC 3041 3042 2157-2175 CUGAGUGCAACUUCUGCAG CUGCAGAAGUUGCACUCAG 3043 3044 2158-2176 UGAGUGCAACUUCUGCAGG CCUGCAGAAGUUGCACUCA 3045 3046 2159-2177 GAGUGCAACUUCUGCAGGA UCCUGCAGAAGUUGCACUC 3047 3048 2160-2178 AGUGCAACUUCUGCAGGAG CUCCUGCAGAAGUUGCACU 3049 3050 2161-2179 GUGCAACUUCUGCAGGAGG CCUCCUGCAGAAGUUGCAC 3051 3052 2162-2180 UGCAACUUCUGCAGGAGGC GCCUCCUGCAGAAGUUGCA 3053 3054 2163-2181 GCAACUUCUGCAGGAGGCC GGCCUCCUGCAGAAGUUGC 3055 3056 2164-2182 CAACUUCUGCAGGAGGCCA UGGCCUCCUGCAGAAGUUG 3057 3058 2165-2183 AACUUCUGCAGGAGGCCAC GUGGCCUCCUGCAGAAGUU 3059 3060 2166-2184 ACUUCUGCAGGAGGCCACU AGUGGCCUCCUGCAGAAGU 3061 3062 2167-2185 CUUCUGCAGGAGGCCACUG CAGUGGCCUCCUGCAGAAG 3063 3064 2168-2186 UUCUGCAGGAGGCCACUGC GCAGUGGCCUCCUGCAGAA 3065 3066 2169-2187 UCUGCAGGAGGCCACUGCA UGCAGUGGCCUCCUGCAGA 3067 3068 2170-2188 CUGCAGGAGGCCACUGCAU AUGCAGUGGCCUCCUGCAG 3069 3070 2171-2189 UGCAGGAGGCCACUGCAUU AAUGCAGUGGCCUCCUGCA 3071 3072 2172-2190 GCAGGAGGCCACUGCAUUU AAAUGCAGUGGCCUCCUGC 3073 3074 2173-2191 CAGGAGGCCACUGCAUUUU AAAAUGCAGUGGCCUCCUG 3075 3076 2174-2192 AGGAGGCCACUGCAUUUUG CAAAAUGCAGUGGCCUCCU 3077 3078 2175-2193 GGAGGCCACUGCAUUUUGA UCAAAAUGCAGUGGCCUCC 3079 3080 2176-2194 GAGGCCACUGCAUUUUGAA UUCAAAAUGCAGUGGCCUC 3081 3082 2177-2195 AGGCCACUGCAUUUUGAAG CUUCAAAAUGCAGUGGCCU 3083 3084 2178-2196 GGCCACUGCAUUUUGAAGU ACUUCAAAAUGCAGUGGCC 3085 3086 2179-2197 GCCACUGCAUUUUGAAGUG CACUUCAAAAUGCAGUGGC 3087 3088 2180-2198 CCACUGCAUUUUGAAGUGA UCACUUCAAAAUGCAGUGG 3089 3090 2181-2199 CACUGCAUUUUGAAGUGAU AUCACUUCAAAAUGCAGUG 3091 3092 2182-2200 ACUGCAUUUUGAAGUGAUG CAUCACUUCAAAAUGCAGU 3093 3094 2183-2201 CUGCAUUUUGAAGUGAUGA UCAUCACUUCAAAAUGCAG 3095 3096 2184-2202 UGCAUUUUGAAGUGAUGAG CUCAUCACUUCAAAAUGCA 3097 3098 2185-2203 GCAUUUUGAAGUGAUGAGU ACUCAUCACUUCAAAAUGC 3099 3100 2186-2204 CAUUUUGAAGUGAUGAGUG CACUCAUCACUUCAAAAUG 3101 3102 2187-2205 AUUUUGAAGUGAUGAGUGA UCACUCAUCACUUCAAAAU 3103 3104 2188-2206 UUUUGAAGUGAUGAGUGAA UUCACUCAUCACUUCAAAA 3105 3106 2190-2208 UUGAAGUGAUGAGUGAAAG CUUUCACUCAUCACUUCAA 3107 3108 2191-2209 UGAAGUGAUGAGUGAAAGA UCUUUCACUCAUCACUUCA 3109 3110 2192-2210 GAAGUGAUGAGUGAAAGAG CUCUUUCACUCAUCACUUC 3111 3112 2193-2211 AAGUGAUGAGUGAAAGAGA UCUCUUUCACUCAUCACUU 3113 3114 2194-2212 AGUGAUGAGUGAAAGAGAG CUCUCUUUCACUCAUCACU 3115 3116 2195-2213 GUGAUGAGUGAAAGAGAGA UCUCUCUUUCACUCAUCAC 3117 3118 2196-2214 UGAUGAGUGAAAGAGAGAA UUCUCUCUUUCACUCAUCA 3119 3120 2197-2215 GAUGAGUGAAAGAGAGAAG CUUCUCUCUUUCACUCAUC 3121 3122 2198-2216 AUGAGUGAAAGAGAGAAGU ACUUCUCUCUUUCACUCAU 3123 3124 2199-2217 UGAGUGAAAGAGAGAAGUC GACUUCUCUCUUUCACUCA 3125 3126 2200-2218 GAGUGAAAGAGAGAAGUCC GGACUUCUCUCUUUCACUC 3127 3128 2201-2219 AGUGAAAGAGAGAAGUCCU AGGACUUCUCUCUUUCACU 3129 3130 2202-2220 GUGAAAGAGAGAAGUCCUA UAGGACUUCUCUCUUUCAC 3131 3132 2203-2221 UGAAAGAGAGAAGUCCUAU AUAGGACUUCUCUCUUUCA 3133 3134 2204-2222 GAAAGAGAGAAGUCCUAUU AAUAGGACUUCUCUCUUUC 3135 3136 2205-2223 AAAGAGAGAAGUCCUAUUU AAAUAGGACUUCUCUCUUU 3137 3138 2206-2224 AAGAGAGAAGUCCUAUUUC GAAAUAGGACUUCUCUCUU 3139 3140 2207-2225 AGAGAGAAGUCCUAUUUCU AGAAAUAGGACUUCUCUCU 3141 3142 2208-2226 GAGAGAAGUCCUAUUUCUC GAGAAAUAGGACUUCUCUC 3143 3144 2209-2227 AGAGAAGUCCUAUUUCUCA UGAGAAAUAGGACUUCUCU 3145 3146 2210-2228 GAGAAGUCCUAUUUCUCAG CUGAGAAAUAGGACUUCUC 3147 3148 2211-2229 AGAAGUCCUAUUUCUCAGG CCUGAGAAAUAGGACUUCU 3149 3150 2212-2230 GAAGUCCUAUUUCUCAGGC GCCUGAGAAAUAGGACUUC 3151 3152 2213-2231 AAGUCCUAUUUCUCAGGCU AGCCUGAGAAAUAGGACUU 3153 3154 2214-2232 AGUCCUAUUUCUCAGGCUU AAGCCUGAGAAAUAGGACU 3155 3156 2215-2233 GUCCUAUUUCUCAGGCUUG CAAGCCUGAGAAAUAGGAC 3157 3158 2216-2234 UCCUAUUUCUCAGGCUUGA UCAAGCCUGAGAAAUAGGA 3159 3160 2217-2235 CCUAUUUCUCAGGCUUGAG CUCAAGCCUGAGAAAUAGG 3161 3162 2218-2236 CUAUUUCUCAGGCUUGAGC GCUCAAGCCUGAGAAAUAG 3163 3164 2219-2237 UAUUUCUCAGGCUUGAGCA UGCUCAAGCCUGAGAAAUA 3165 3166 2220-2238 AUUUCUCAGGCUUGAGCAA UUGCUCAAGCCUGAGAAAU 3167 3168 2221-2239 UUUCUCAGGCUUGAGCAAG CUUGCUCAAGCCUGAGAAA 3169 3170 2222-2240 UUCUCAGGCUUGAGCAAGU ACUUGCUCAAGCCUGAGAA 3171 3172 2223-2241 UCUCAGGCUUGAGCAAGUU AACUUGCUCAAGCCUGAGA 3173 3174 2224-2242 CUCAGGCUUGAGCAAGUUG CAACUUGCUCAAGCCUGAG 3175 3176 2225-2243 UCAGGCUUGAGCAAGUUGG CCAACUUGCUCAAGCCUGA 3177 3178 2226-2244 CAGGCUUGAGCAAGUUGGU ACCAACUUGCUCAAGCCUG 3179 3180 2229-2247 GCUUGAGCAAGUUGGUAUC GAUACCAACUUGCUCAAGC 3181 3182 2231-2249 UUGAGCAAGUUGGUAUCUG CAGAUACCAACUUGCUCAA 3183 3184 2232-2250 UGAGCAAGUUGGUAUCUGC GCAGAUACCAACUUGCUCA 3185 3186 2233-2251 GAGCAAGUUGGUAUCUGCU AGCAGAUACCAACUUGCUC 3187 3188 2234-2252 AGCAAGUUGGUAUCUGCUC GAGCAGAUACCAACUUGCU 3189 3190 2235-2253 GCAAGUUGGUAUCUGCUCA UGAGCAGAUACCAACUUGC 3191 3192 2236-2254 CAAGUUGGUAUCUGCUCAG CUGAGCAGAUACCAACUUG 3193 3194 2237-2255 AAGUUGGUAUCUGCUCAGG CCUGAGCAGAUACCAACUU 3195 3196 2238-2256 AGUUGGUAUCUGCUCAGGC GCCUGAGCAGAUACCAACU 3197 3198 2239-2257 GUUGGUAUCUGCUCAGGCC GGCCUGAGCAGAUACCAAC 3199 3200 2240-2258 UUGGUAUCUGCUCAGGCCU AGGCCUGAGCAGAUACCAA 3201 3202 2241-2259 UGGUAUCUGCUCAGGCCUG CAGGCCUGAGCAGAUACCA 3203 3204 2242-2260 GGUAUCUGCUCAGGCCUGA UCAGGCCUGAGCAGAUACC 3205 3206 2243-2261 GUAUCUGCUCAGGCCUGAG CUCAGGCCUGAGCAGAUAC 3207 3208 2244-2262 UAUCUGCUCAGGCCUGAGC GCUCAGGCCUGAGCAGAUA 3209 3210 2245-2263 AUCUGCUCAGGCCUGAGCA UGCUCAGGCCUGAGCAGAU 3211 3212 2246-2264 UCUGCUCAGGCCUGAGCAU AUGCUCAGGCCUGAGCAGA 3213 3214 2247-2265 CUGCUCAGGCCUGAGCAUG CAUGCUCAGGCCUGAGCAG 3215 3216 2248-2266 UGCUCAGGCCUGAGCAUGA UCAUGCUCAGGCCUGAGCA 3217 3218 2249-2267 GCUCAGGCCUGAGCAUGAC GUCAUGCUCAGGCCUGAGC 3219 3220 2250-2268 CUCAGGCCUGAGCAUGACC GGUCAUGCUCAGGCCUGAG 3221 3222 2251-2269 UCAGGCCUGAGCAUGACCU AGGUCAUGCUCAGGCCUGA 3223 3224 2252-2270 CAGGCCUGAGCAUGACCUC GAGGUCAUGCUCAGGCCUG 3225 3226 2253-2271 AGGCCUGAGCAUGACCUCA UGAGGUCAUGCUCAGGCCU 3227 3228 2279-2297 CACUUAACCCCAGGCCAUU AAUGGCCUGGGGUUAAGUG 3229 3230 2280-2298 ACUUAACCCCAGGCCAUUA UAAUGGCCUGGGGUUAAGU 3231 3232 2281-2299 CUUAACCCCAGGCCAUUAU AUAAUGGCCUGGGGUUAAG 3233 3234 2282-2300 UUAACCCCAGGCCAUUAUC GAUAAUGGCCUGGGGUUAA 3235 3236 2283-2301 UAACCCCAGGCCAUUAUCA UGAUAAUGGCCUGGGGUUA 3237 3238 2284-2302 AACCCCAGGCCAUUAUCAU AUGAUAAUGGCCUGGGGUU 3239 3240 2285-2303 ACCCCAGGCCAUUAUCAUA UAUGAUAAUGGCCUGGGGU 3241 3242 2287-2305 CCCAGGCCAUUAUCAUAUC GAUAUGAUAAUGGCCUGGG 3243 3244 2288-2306 CCAGGCCAUUAUCAUAUCC GGAUAUGAUAAUGGCCUGG 3245 3246 2289-2307 CAGGCCAUUAUCAUAUCCA UGGAUAUGAUAAUGGCCUG 3247 3248 2290-2308 AGGCCAUUAUCAUAUCCAG CUGGAUAUGAUAAUGGCCU 3249 3250 2291-2309 GGCCAUUAUCAUAUCCAGA UCUGGAUAUGAUAAUGGCC 3251 3252 2292-2310 GCCAUUAUCAUAUCCAGAU AUCUGGAUAUGAUAAUGGC 3253 3254 2314-2332 CUUCAGAGUUGUCUUUAUA UAUAAAGACAACUCUGAAG 3255 3256 2315-2333 UUCAGAGUUGUCUUUAUAU AUAUAAAGACAACUCUGAA 3257 3258 2316-2334 UCAGAGUUGUCUUUAUAUG CAUAUAAAGACAACUCUGA 3259 3260 2318-2336 AGAGUUGUCUUUAUAUGUG CACAUAUAAAGACAACUCU 3261 3262 2322-2340 UUGUCUUUAUAUGUGAAUU AAUUCACAUAUAAAGACAA 3263 3264 2323-2341 UGUCUUUAUAUGUGAAUUA UAAUUCACAUAUAAAGACA 3265 3266 2324-2342 GUCUUUAUAUGUGAAUUAA UUAAUUCACAUAUAAAGAC 3267 3268 2325-2343 UCUUUAUAUGUGAAUUAAG CUUAAUUCACAUAUAAAGA 3269 3270 2326-2344 CUUUAUAUGUGAAUUAAGU ACUUAAUUCACAUAUAAAG 3271 3272 2327-2345 UUUAUAUGUGAAUUAAGUU AACUUAAUUCACAUAUAAA 3273 3274 2328-2346 UUAUAUGUGAAUUAAGUUA UAACUUAAUUCACAUAUAA 3275 3276 2329-2347 UAUAUGUGAAUUAAGUUAU AUAACUUAAUUCACAUAUA 3277 3278 2330-2348 AUAUGUGAAUUAAGUUAUA UAUAACUUAAUUCACAUAU 3279 3280 2331-2349 UAUGUGAAUUAAGUUAUAU AUAUAACUUAAUUCACAUA 3281 3282 2332-2350 AUGUGAAUUAAGUUAUAUU AAUAUAACUUAAUUCACAU 3283 3284 2333-2351 UGUGAAUUAAGUUAUAUUA UAAUAUAACUUAAUUCACA 3285 3286 2334-2352 GUGAAUUAAGUUAUAUUAA UUAAUAUAACUUAAUUCAC 3287 3288 2335-2353 UGAAUUAAGUUAUAUUAAA UUUAAUAUAACUUAAUUCA 3289 3290 2336-2354 GAAUUAAGUUAUAUUAAAU AUUUAAUAUAACUUAAUUC 3291 3292 2337-2355 AAUUAAGUUAUAUUAAAUU AAUUUAAUAUAACUUAAUU 3293 3294 2338-2356 AUUAAGUUAUAUUAAAUUU AAAUUUAAUAUAACUUAAU 3295 3296 2339-2357 UUAAGUUAUAUUAAAUUUU AAAAUUUAAUAUAACUUAA 3297 3298 2340-2358 UAAGUUAUAUUAAAUUUUA UAAAAUUUAAUAUAACUUA 3299 3300 2341-2359 AAGUUAUAUUAAAUUUUAA UUAAAAUUUAAUAUAACUU 3301 3302 2342-2360 AGUUAUAUUAAAUUUUAAU AUUAAAAUUUAAUAUAACU 3303 3304 2343-2361 GUUAUAUUAAAUUUUAAUC GAUUAAAAUUUAAUAUAAC 3305 3306 2345-2363 UAUAUUAAAUUUUAAUCUA UAGAUUAAAAUUUAAUAUA 3307 3308 2346-2364 AUAUUAAAUUUUAAUCUAU AUAGAUUAAAAUUUAAUAU 3309 3310 2347-2365 UAUUAAAUUUUAAUCUAUA UAUAGAUUAAAAUUUAAUA 3311 3312 2348-2366 AUUAAAUUUUAAUCUAUAG CUAUAGAUUAAAAUUUAAU 3313 3314 2349-2367 UUAAAUUUUAAUCUAUAGU ACUAUAGAUUAAAAUUUAA 3315 3316 2350-2368 UAAAUUUUAAUCUAUAGUA UACUAUAGAUUAAAAUUUA 3317 3318 2351-2369 AAAUUUUAAUCUAUAGUAA UUACUAUAGAUUAAAAUUU 3319 3320 2354-2372 UUUUAAUCUAUAGUAAAAA UUUUUACUAUAGAUUAAAA 3321 3322 2355-2373 UUUAAUCUAUAGUAAAAAC GUUUUUACUAUAGAUUAAA 3323 3324 2356-2374 UUAAUCUAUAGUAAAAACA UGUUUUUACUAUAGAUUAA 3325 3326 2357-2375 UAAUCUAUAGUAAAAACAU AUGUUUUUACUAUAGAUUA 3327 3328 2358-2376 AAUCUAUAGUAAAAACAUA UAUGUUUUUACUAUAGAUU 3329 3330 2359-2377 AUCUAUAGUAAAAACAUAG CUAUGUUUUUACUAUAGAU 3331 3332 2360-2378 UCUAUAGUAAAAACAUAGU ACUAUGUUUUUACUAUAGA 3333 3334 2361-2379 CUAUAGUAAAAACAUAGUC GACUAUGUUUUUACUAUAG 3335 3336 2362-2380 UAUAGUAAAAACAUAGUCC GGACUAUGUUUUUACUAUA 3337 3338 2363-2381 AUAGUAAAAACAUAGUCCU AGGACUAUGUUUUUACUAU 3339 3340 2364-2382 UAGUAAAAACAUAGUCCUG CAGGACUAUGUUUUUACUA 3341 3342 2365-2383 AGUAAAAACAUAGUCCUGG CCAGGACUAUGUUUUUACU 3343 3344 2366-2384 GUAAAAACAUAGUCCUGGA UCCAGGACUAUGUUUUUAC 3345 3346 2367-2385 UAAAAACAUAGUCCUGGAA UUCCAGGACUAUGUUUUUA 3347 3348 2368-2386 AAAAACAUAGUCCUGGAAA UUUCCAGGACUAUGUUUUU 3349 3350 2369-2387 AAAACAUAGUCCUGGAAAU AUUUCCAGGACUAUGUUUU 3351 3352 2370-2388 AAACAUAGUCCUGGAAAUA UAUUUCCAGGACUAUGUUU 3353 3354 2371-2389 AACAUAGUCCUGGAAAUAA UUAUUUCCAGGACUAUGUU 3355 3356 2372-2390 ACAUAGUCCUGGAAAUAAA UUUAUUUCCAGGACUAUGU 3357 3358 2373-2391 CAUAGUCCUGGAAAUAAAU AUUUAUUUCCAGGACUAUG 3359 3360 2374-2392 AUAGUCCUGGAAAUAAAUU AAUUUAUUUCCAGGACUAU 3361 3362 2375-2393 UAGUCCUGGAAAUAAAUUC GAAUUUAUUUCCAGGACUA 3363 3364 2377-2395 GUCCUGGAAAUAAAUUCUU AAGAAUUUAUUUCCAGGAC 3365 3366 2378-2396 UCCUGGAAAUAAAUUCUUG CAAGAAUUUAUUUCCAGGA

Example 9. Suppression of Porphyrin Precursors Using ALAS1 siRNA in an Acute Treatment Paradigm

The AIP mouse model (see Example 5) was used to investigate whether ALAS1 siRNA would work an an acute treatment paradigm to lower already elevated levels of ALA and PBG, as would be present, for example, when a human porphyria patient suffers from an acute attack. Administration of the AD-53558 LNP11 formulation siRNA at a 1 mg/kg dose 12 hours after the last dose of phenobarbitol rapidly decreased the levels of both ALA and PBG in mouse plasma, whereas in Luc control treated animals the levels continued to rise (FIG. 14). These results indicate that ALAS siRNA is effective for treating an acute attack. The ALAS1 siRNA was effective to lower and prevent further increases in ALA and PBG levels.

Example 10. siRNAs that Target ALAS1

Further unmodified and modified siRNA sequences that target ALAS1 siRNA were designed and produced as described in Example 2. The in vitro activity of the modified duplexes was tested as described below.

Methods Lipid Mediated Transfection

For Hep3B, PMH, and primary Cynomolgus hepatocytes, transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. catalog number 13778-150) to 5 μl of each siRNA duplex to an individual well in a 96-well plate. The mixture was then incubated at room temperature for 20 minutes. Eighty μl of complete growth media without antibiotic containing the appropriate cell number were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification.

Single dose experiments were performed at 1 uM, 500 nM, 20 nM, 10 nM and 0.2 nM final duplex concentration for GalNAc modified.

Free Uptake Transfection

Cryopreserved Primary Cynomolgus Hepatocytes (Celsius In Vitro Technologies, MO03055-P) were thawed at 37° C. water bath immediately prior to usage and re-suspended at 0.26×10⁶ cells/ml in InVitroGRO CP (plating) medium (Celsius In Vitro Technologies, catalog number Z99029). During transfections, cells were plated onto a BD BioCoat 96 well collagen plate (BD, 356407) at 25,000 cells per well and incubated at 37° C. in an atmosphere of 5% CO₂. Free Uptake experiments were performed by adding 10 μl of siRNA duplexes in PBS per well into a 96 well (96w) plate. Ninety μl of complete growth media containing appropriate cell number for the cell type was then added to the siRNA. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 1 uM, 500 nM, 20 nM and 10 nM final duplex.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12)

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using a magnetic stand and the supernatant was removed without disturbing the beads. After removing the supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing the supernatant, magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixed for 1 minute. The beads were captured again and the supernatant was removed. The beads were then washed with 150 μl Wash Buffer B, captured and the supernatant was removed. The beads were next washed with 150 μl Elution Buffer, captured and the supernatant removed. Finally, the beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. The beads were captured on magnet for 5 minutes. Forty-five μl of supernatant was removed and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction as prepared. Equal volumes master mix and RNA were mixed for a final volume of 12 μl for in vitro screened or 20 μl for in vivo screened samples. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. for 10 minutes, 37° C. for 120 minutes, 85° C. for 5 seconds, and 4° C. hold.

Real time PCR

Two μl of cDNA were added to a master mix containing 2 μl of H₂O, 0.5 μl GAPDH TaqMan Probe (Life Technologies catalog number 4326317E for Hep3B cells, catalog number 352339E for primary mouse hepatocytes or custom probe for cynomolgus primary hepatocytes), 0.5 μl C5 TaqMan probe (Life Technologies catalog number Hs00167441_m1 for Hep3B cells or Mm00457879_m1 for Primary Mouse Hepatoctyes or custom probe for cynomolgus primary hepatocytes) and 5 μl Lightcycler 480 probe master mix (Roche catalog number 04887301001) per well in a 384 well (384 w) plates (Roche catalog number 04887301001). Real time PCR was performed in an Roche LC480 Real Time PCR system (Roche) using the ΔΔCt(RQ) assay. For in vitro screening, each duplex was tested with two biological replicates unless otherwise noted and each Real Time PCR was performed in duplicate technical replicates. For in vivo screening, each duplex was tested in one or more experiments (3 mice per group) and each Real Time PCR was run in duplicate technical replicates.

To calculate relative fold change in ALAS1 mRNA levels, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC₅₀s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 over the same dose range, or to its own lowest dose.

The sense and antisense sequences of AD-1955 are:

SENSE: (SEQ ID NO: 3682) cuuAcGcuGAGuAcuucGAdTsdT ANTISENSE: (SEQ ID NO: 3683) UCGAAGuACUcAGCGuAAGdTsdT.

The single strand and duplex sequences of the modified and unmodified siRNAs are provided in Table 14 and Table 15, respectively.

TABLE 14 Human ALAS1 Modified Single Strands and Duplex Sequences SEQ Target ID sites of SEQ ID NO: antisense NO: (anti- Duplex sequence on (sense) sense) Name Sense Sequence (5′-3′) Antisense Sequence (5′-3′) NM_000688.4 3371 3372 AD-58848 CfsasUfgCfcAfaAfAfAfuGfgAfcA asUfsgAfuGfuCfcAfuuuUfuGfgCfaU 1635-1657 fuCfaUfL96 fgsAfsc 3373 3374 AD-58849 AfsusUfuUfgAfaGfUfGfaUfgAfgU usUfsuCfaCfuCfaUfcacUfuCfaAfaA 2189-2211 fgAfaAfL96 fusGfsc 3375 3376 AD-58850 AfsgsUfuAfuAfuUfAfAfaUfuUfuA asGfsaUfuAfaAfaUfuuaAfuAfuAfaC 2344-2366 faUfcUfL96 fusUfsa 3377 3378 AD-58851 GfscsAfuUfuUfgAfAfGfuGfaUfgA usCfsaCfuCfaUfcAfcuuCfaAfaAfuG 2187-2209 fgUfgAfL96 fcsAfsg 3379 3380 AD-58852 GfsasAfcUfaAfuGfAfGfcAfgAfcA gsUfsuAfuGfuCfuGfcucAfuUfaGfuU 1975-1997 fuAfaCfL96 fcsAfsu 3381 3382 AD-58853 AfsasUfgAfcCfaCfAfCfcUfaUfcG asAfscUfcGfaUfaGfgugUfgGfuCfaU 973-995 faGfuUfL96 fusCfsu 3383 3384 AD-58854 UfsasAfaUfuUfuAfAfUfcUfaUfaG usUfsuAfcUfaUfaGfauuAfaAfaUfuU 2352-2374 fuAfaAfL96 fasAfsu 3385 3386 AD-58855 UfsusCfaGfuAfuGfAfUfcGfuUfuC csAfsaAfgAfaAfcGfaucAfuAfcUfgA 929-951 fuUfuGfL96 fasAfsa 3387 3388 AD-58856 CfsasCfuUfuUfcAfGfUfaUfgAfuC asAfsaCfgAfuCfaUfacuGfaAfaAfgU 924-946 fgUfuUfL96 fgsGfsa 3389 3390 AD-58857 AfsasAfuCfuGfuUfUfCfcAfcUfuU csUfsgAfaAfaGfuGfgaaAfcAfgAfuU 913-935 fuCfaGfL96 fusUfsg 3391 3392 AD-58858 CfsasUfuUfgAfaAfCfUfgUfcCfaU usUfsgAfaUfgGfaCfaguUfuCfaAfaU 1478-1500 fuCfaAfL96 fgsCfsc 3393 3394 AD-58859 CfscsUfaUfcGfaGfUfUfuUfuAfaA csAfsgUfuUfuAfaAfaacUfcGfaUfaG  983-1005 faCfuGfL96 fgsUfsg 3395 3396 AD-58861 GfsasCfcAfgAfaAfGfAfgUfgUfcU gsAfsuGfaGfaCfaCfucuUfuCfuGfgU 872-894 fcAfuCfL96 fcsUfsu 3397 3398 AD-58862 AfscsCfaGfaAfaGfAfGfuGfuCfuC asGfsaUfgAfgAfcAfcucUfuUfcUfgG 873-895 faUfcUfL96 fusCfsu 3399 3400 AD-58863 AfscsUfaAfuGfaGfCfAfgAfcAfuA asUfsgUfuAfuGfuCfugcUfcAfuUfaG 1977-1999 faCfaUfL96 fusUfsc 3401 3402 AD-58864 UfsasGfuAfaAfaAfCfAfuAfgUfcC usCfscAfgGfaCfuAfuguUfuUfuAfcU 2366-2388 fuGfgAfL96 fasUfsa 3403 3404 AD-58865 UfsasUfuUfcUfgGfAfAfcUfaGfuA asAfsuUfuAfcUfaGfuucCfaGfaAfaU 1185-1207 faAfuUfL96 fasUfsu 3405 3406 AD-58867 UfsusCfuGfcAfaAfGfCfcAfgUfcU csUfscAfaGfaCfuGfgcuUfuGfcAfgA 706-728 fuGfaGfL96 fasGfsa 3407 3408 AD-58868 GfsasGfgAfaAfgAfGfGfuUfgCfuG gsUfsuUfcAfgCfaAfccuCfuUfuCfcU 759-781 faAfaCfL96 fcsAfsc 3409 3410 AD-58869 GfsgsUfaCfuAfgAfAfAfuAfuUfuC usCfscAfgAfaAfuAfuuuCfuAfgUfaC 1174-1196 fuGfgAfL96 fcsAfsc 3411 3412 AD-58870 GfsasCfaUfcAfuGfCfAfaAfaGfcA usCfsuUfuGfcUfuUfugcAfuGfaUfgU 853-875 faAfgAfL96 fcsCfsu 3413 3414 AD-58871 AfsasAfuUfuUfaAfUfCfuAfuAfgU usUfsuUfaCfuAfuAfgauUfaAfaAfuU 2353-2375 faAfaAfL96 fusAfsa 3415 3416 AD-58873 CfsasUfgAfuCfcAfAfGfgGfaUfuC usUfsuCfgAfaUfcCfcuuGfgAfuCfaU 1362-1384 fgAfaAfL96 fgsGfsa 3417 3418 AD-58874 AfsgsAfcCfaGfaAfAfGfaGfuGfuC asUfsgAfgAfcAfcUfcuuUfcUfgGfuC 871-893 fuCfaUfL96 fusUfsu 3419 3420 AD-58875 AfsusCfcUfgAfaGfAfGfcGfcUfgA usCfscCfuCfaGfcGfcucUfuCfaGfgA 1810-1832 fgGfgAfL96 fusCfsc 3421 3422 AD-58876 GfsusCfuGfuGfaUfGfAfaCfuAfaU gsCfsuCfaUfuAfgUfucaUfcAfcAfgA 1966-1988 fgAfgCfL96 fcsUfsu 3423 3424 AD-58877 CfsasGfaAfaGfaGfUfGfuCfuCfaU gsAfsaGfaUfgAfgAfcacUfcUfuUfcU 875-897 fcUfuCfL96 fgsGfsu 3425 3426 AD-58878 AfscsUfuUfuCfaGfUfAfuGfaUfcG gsAfsaAfcGfaUfcAfuacUfgAfaAfaG 925-947 fuUfuCfL96 fusGfsg 3427 3428 AD-58879 UfscsAfuGfcCfaAfAfAfaUfgGfaC usGfsaUfgUfcCfaUfuuuUfgGfcAfuG 1634-1656 faUfcAfL96 fasCfsu 3429 3430 AD-58880 AfsasUfaUfuUfcUfGfGfaAfcUfaG usUfsuAfcUfaGfuUfccaGfaAfaUfaU 1183-1205 fuAfaAfL96 fusUfsc 3431 3432 AD-58881 CfsusUfcUfuCfaAfGfAfuAfaCfuU usGfsgCfaAfgUfuAfucuUfgAfaGfaA 892-914 fgCfcAfL96 fgsAfsu 3433 3434 AD-58882 UfsusUfcAfgUfaUfGfAfuCfgUfuU asAfsaGfaAfaCfgAfucaUfaCfuGfaA 928-950 fcUfuUfL96 fasAfsg 3435 3436 AD-58883 CfscsCfaGfuGfuGfGfUfuAfgUfgU usUfsuCfaCfaCfuAfaccAfcAfcUfgG 790-812 fgAfaAfL96 fgsGfsc 3437 3438 AD-58884 GfscsUfgUfgAfgAfUfUfuAfcUfcU asAfsuCfaGfaGfuAfaauCfuCfaCfaG 1325-1347 fgAfuUfL96 fcsCfsu 3439 3440 AD-58885 AfsgsGfcUfuGfaGfCfAfaGfuUfgG gsAfsuAfcCfaAfcUfugcUfcAfaGfcC 2229-2251 fuAfuCfL96 fusGfsa 3441 3442 AD-58886 GfsasAfaGfaGfuGfUfCfuCfaUfcU asAfsgAfaGfaUfgAfgacAfcUfcUfuU 877-899 fuCfuUfL96 fcsUfsg 3443 3444 AD-58887 AfsusUfuCfuGfgAfAfCfuAfgUfaA gsAfsaUfuUfaCfuAfguuCfcAfgAfaA 1186-1208 faUfuCfL96 fusAfsu 3445 3446 AD-58888 UfsgsUfgAfuGfuGfGfCfcCfaUfgA asAfsaCfuCfaUfgGfgccAfcAfuCfaC 1531-1553 fgUfuUfL96 fasCfsa 3447 3448 AD-58889 AfsasGfaGfaGfaAfGfUfcCfuAfuU gsAfsgAfaAfuAfgGfacuUfcUfcUfcU 2208-2230 fuCfuCfL96 fusUfsc 3449 3450 AD-58890 UfsgsGfcAfgCfaCfAfGfaUfgAfaU usCfsuGfaUfuCfaUfcugUfgCfuGfcC 671-693 fcAfgAfL96 fasGfsg 3451 3452 AD-58891 AfsusGfaUfcGfuUfUfCfuUfuGfaG usUfsuUfcUfcAfaAfgaaAfcGfaUfcA 935-957 faAfaAfL96 fusAfsc 3453 3454 AD-58892 UfscsUfgGfaAfcUfAfGfuAfaAfuU asUfsgGfaAfuUfuAfcuaGfuUfcCfaG 1189-1211 fcCfaUfL96 fasAfsa 3455 3456 AD-59095 GfscsCfcAfuUfcUfUfAfuCfcCfgA asCfsuCfgGfgAfuAfagaAfuGfgsgsc 360-382 fgUfL96 3457 3458 AD-59096 GfsgsAfaCfcAfuGfCfCfuCfcAfuG asUfscAfuGfgAfgGfcauGfgUfuscsc 1347-1369 faUfL96 3459 3460 AD-59097 UfsgsGfaGfuCfuGfUfGfcGfgAfuC asGfsgAfuCfcGfcAfcagAfcUfcscsa 1794-1816 fcUfL96 3461 3462 AD-59098 CfsasCfcCfaCfgGfGfUfgUfgUfgG usCfscCfaCfaCfaCfccgUfgGfgsusg 1112-1134 fgAfL96 3463 3464 AD-59099 GfsgsAfgUfcUfgUfGfCfgGfaUfcC usAfsgGfaUfcCfgCfacaGfaCfuscsc 1795-1817 fuAfL96 3465 3466 AD-59100 CfsasAfaAfcUfgCfCfCfcAfaGfaU usCfsaUfcUfuGfgGfgcaGfuUfususg 428-450 fgAfL96 3467 3468 AD-59101 GfscsCfuCfcAfuGfAfUfcCfaAfgG usCfscCfuUfgGfaUfcauGfgAfgsgsc 1355-1377 fgAfL96 3469 3470 AD-59102 CfsasUfcAfuCfcCfUfGfuGfcGfgG asAfscCfcGfcAfcAfgggAfuGfasusg 1921-1943 fuUfL96 3471 3472 AD-59103 AfscsCfcAfcGfgGfUfGfuGfuGfgG usCfscCfcAfcAfcAfcccGfuGfgsgsu 1113-1135 fgAfL96 3473 3474 AD-59104 CfsasCfaUfcAfuCfCfCfuGfuGfcG usCfscGfcAfcAfgGfgauGfaUfgsusg 1919-1941 fgAfL96 3475 3476 AD-59105 CfsasGfaAfaGfaGfUfGfuCfuCfaU asGfsaUfgAfgAfcAfcucUfuUfcsusg 873-895 fcUfL96 3477 3478 AD-59106 CfscsUfcCfaUfgAfUfCfcAfaGfgG asUfscCfcUfuGfgAfucaUfgGfasgsg 1356-1378 faUfL96 3479 3480 AD-59107 UfsgsCfcCfaUfuCfUfUfaUfcCfcG usUfscGfgGfaUfaAfgaaUfgGfgscsa 359-381 faAfL96 3481 3482 AD-59108 CfsusUfcAfcCfcUfGfGfcUfaAfgA usAfsuCfuUfaGfcCfaggGfuGfasasg 1297-1319 fuAfL96 3483 3484 AD-59109 AfsusCfaUfcCfcUfGfUfgCfgGfgU usAfsaCfcCfgCfaCfaggGfaUfgsasu 1922-1944 fuAfL96 3485 3486 AD-59110 AfsgsAfaAfgAfgUfGfUfcUfcAfuC asAfsgAfuGfaGfaCfacuCfuUfuscsu 874-896 fuUfL96 3487 3488 AD-59111 CfsusCfcAfuGfaUfCfCfaAfgGfgA asAfsuCfcCfuUfgGfaucAfuGfgsasg 1357-1379 fuUfL96 3489 3490 AD-59112 CfscsAfuUfcUfuAfUfCfcCfgAfgU usGfsaCfuCfgGfgAfuaaGfaAfusgsg 362-384 fcAfL96 3491 3492 AD-59113 CfsasCfcCfuGfgCfUfAfaGfaUfgA usAfsuCfaUfcUfuAfgccAfgGfgsusg 1300-1322 fuAfL96 3493 3494 AD-59114 UfscsAfuCfcCfuGfUfGfcGfgGfuU usCfsaAfcCfcGfcAfcagGfgAfusgsa 1923-1945 fgAfL96 3495 3496 AD-59115 AfsasGfaGfuGfuCfUfCfaUfcUfuC asAfsgAfaGfaUfgAfgacAfcUfcsusu 877-899 fuUfL96 3497 3498 AD-59116 GfsusCfaUfgCfcAfAfAfaAfuGfgA usGfsuCfcAfuUfuUfuggCfaUfgsasc 1631-1653 fcAfL96 3499 3500 AD-59117 CfsasUfuCfuUfaUfCfCfcGfaGfuC usGfsgAfcUfcGfgGfauaAfgAfasusg 363-385 fcAfL96 3501 3502 AD-59118 AfscsCfcUfgGfcUfAfAfgAfuGfaU usCfsaUfcAfuCfuUfagcCfaGfgsgsu 1301-1323 fgAfL96 3503 3504 AD-59119 CfsusCfuUfcAfcCfCfUfgGfcUfaA usCfsuUfaGfcCfaGfgguGfaAfgsasg 1295-1317 fgAfL96 3505 3506 AD-59120 AfsusGfcCfaAfaAfAfUfgGfaCfaU usGfsaUfgUfcCfaUfuuuUfgGfcsasu 1634-1656 fcAfL96 3507 3508 AD-59121 UfsgsCfcCfcAfaGfAfUfgAfuGfgA asUfsuCfcAfuCfaUfcuuGfgGfgscsa 434-456 faUfL96 3509 3510 AD-59122 GfsasAfcCfaUfgCfCfUfcCfaUfgA usAfsuCfaUfgGfaGfgcaUfgGfususc 1348-1370 fuAfL96 3511 3512 AD-59123 UfscsUfuCfaCfcCfUfGfgCfuAfaG asUfscUfuAfgCfcAfgggUfgAfasgsa 1296-1318 faUfL96 3513 3514 AD-59124 UfsgsCfcAfaAfaAfUfGfgAfcAfuC asUfsgAfuGfuCfcAfuuuUfuGfgscsa 1635-1657 faUfL96 3515 3516 AD-59125 CfscsAfgAfaAfgAfGfUfgUfcUfcA usAfsuGfaGfaCfaCfucuUfuCfusgsg 872-894 fuAfL96 3517 3518 AD-59126 GfsasAfaCfuGfuCfCfAfuUfcAfaU usCfsaUfuGfaAfuGfgacAfgUfususc 1481-1503 fgAfL96 3519 3520 AD-59127 UfscsAfcCfcUfgGfCfUfaAfgAfuG asUfscAfuCfuUfaGfccaGfgGfusgsa 1299-1321 faUfL96 3521 3522 AD-59128 CfscsCfuGfgAfgUfCfUfgUfgCfgG asUfscCfgCfaCfaGfacuCfcAfgsgsg 1791-1813 faUfL96 3523 3524 AD-59129 GfsasAfaGfaGfuGfUfCfuCfaUfcU usAfsaGfaUfgAfgAfcacUfcUfususc 875-897 fuAfL96 3525 3526 AD-59130 UfsgsGfaGfcCfcUfGfGfaGfuCfuG usAfscAfgAfcUfcCfaggGfcUfcscsa 1786-1808 fuAfL96

TABLE 15 Human ALASl Unmodified Single Strands and Duplex Sequences Target SEQ sites of SEQ ID ID NO: antisense   NO: (anti- Duplex Sense Sequence Antisense Sequence sequence on (sense) sense) Name (5′-3′) (5′-3′) NM_000688.4 3684 3527 AD-58848 CAUGCCAAAAAUGGACAUCAU AUGAUGUCCAUUUUUGGCAUGAC 1635-1657 3528 3529 AD-58849 AUUUUGAAGUGAUGAGUGAAA UUUCACUCAUCACUUCAAAAUGC 2189-2211 3530 3531 AD-58850 AGUUAUAUUAAAUUUUAAUCU AGAUUAAAAUUUAAUAUAACUUA 2344-2366 3532 3533 AD-58851 GCAUUUUGAAGUGAUGAGUGA UCACUCAUCACUUCAAAAUGCAG 2187-2209 3534 3535 AD-58852 GAACUAAUGAGCAGACAUAAC GUUAUGUCUGCUCAUUAGUUCAU 1975-1997 3536 3537 AD-58853 AAUGACCACACCUAUCGAGUU AACUCGAUAGGUGUGGUCAUUCU 973-995 3538 3539 AD-58854 UAAAUUUUAAUCUAUAGUAAA UUUACUAUAGAUUAAAAUUUAAU 2352-2374 3540 3541 AD-58855 UUCAGUAUGAUCGUUUCUUUG CAAAGAAACGAUCAUACUGAAAA 929-951 3542 3543 AD-58856 CACUUUUCAGUAUGAUCGUUU AAACGAUCAUACUGAAAAGUGGA 924-946 3544 3545 AD-58857 AAAUCUGUUUCCACUUUUCAG CUGAAAAGUGGAAACAGAUUUUG 913-935 3546 3547 AD-58858 CAUUUGAAACUGUCCAUUCAA UUGAAUGGACAGUUUCAAAUGCC 1478-1500 3548 3549 AD-58859 CCUAUCGAGUUUUUAAAACUG CAGUUUUAAAAACUCGAUAGGUG  983-1005 3550 3551 AD-58861 GACCAGAAAGAGUGUCUCAUC GAUGAGACACUCUUUCUGGUCUU 872-894 3552 3553 AD-58862 ACCAGAAAGAGUGUCUCAUCU AGAUGAGACACUCUUUCUGGUCU 873-895 3554 3555 AD-58863 ACUAAUGAGCAGACAUAACAU AUGUUAUGUCUGCUCAUUAGUUC 1977-1999 3556 3557 AD-58864 UAGUAAAAACAUAGUCCUGGA UCCAGGACUAUGUUUUUACUAUA 2366-2388 3558 3559 AD-58865 UAUUUCUGGAACUAGUAAAUU AAUUUACUAGUUCCAGAAAUAUU 1185-1207 3560 3561 AD-58867 UUCUGCAAAGCCAGUCUUGAG CUCAAGACUGGCUUUGCAGAAGA 706-728 3562 3563 AD-58868 GAGGAAAGAGGUUGCUGAAAC GUUUCAGCAACCUCUUUCCUCAC 759-781 3564 3565 AD-58869 GGUACUAGAAAUAUUUCUGGA UCCAGAAAUAUUUCUAGUACCAC 1174-1196 3566 3567 AD-58870 GACAUCAUGCAAAAGCAAAGA UCUUUGCUUUUGCAUGAUGUCCU 853-875 3568 3569 AD-58871 AAAUUUUAAUCUAUAGUAAAA UUUUACUAUAGAUUAAAAUUUAA 2353-2375 3570 3571 AD-58873 CAUGAUCCAAGGGAUUCGAAA UUUCGAAUCCCUUGGAUCAUGGA 1362-1384 3572 3573 AD-58874 AGACCAGAAAGAGUGUCUCAU AUGAGACACUCUUUCUGGUCUUU 871-893 3574 3575 AD-58875 AUCCUGAAGAGCGCUGAGGGA UCCCUCAGCGCUCUUCAGGAUCC 1810-1832 3576 3577 AD-58876 GUCUGUGAUGAACUAAUGAGC GCUCAUUAGUUCAUCACAGACUU 1966-1988 3578 3579 AD-58877 CAGAAAGAGUGUCUCAUCUUC GAAGAUGAGACACUCUUUCUGGU 875-897 3580 3581 AD-58878 ACUUUUCAGUAUGAUCGUUUC GAAACGAUCAUACUGAAAAGUGG 925-947 3582 3583 AD-58879 UCAUGCCAAAAAUGGACAUCA UGAUGUCCAUUUUUGGCAUGACU 1634-1656 3584 3585 AD-58880 AAUAUUUCUGGAACUAGUAAA UUUACUAGUUCCAGAAAUAUUUC 1183-1205 3586 3587 AD-58881 CUUCUUCAAGAUAACUUGCCA UGGCAAGUUAUCUUGAAGAAGAU 892-914 3588 3589 AD-58882 UUUCAGUAUGAUCGUUUCUUU AAAGAAACGAUCAUACUGAAAAG 928-950 3590 3591 AD-58883 CCCAGUGUGGUUAGUGUGAAA UUUCACACUAACCACACUGGGGC 790-812 3592 3593 AD-58884 GCUGUGAGAUUUACUCUGAUU AAUCAGAGUAAAUCUCACAGCCU 1325-1347 3594 3595 AD-58885 AGGCUUGAGCAAGUUGGUAUC GAUACCAACUUGCUCAAGCCUGA 2229-2251 3596 3597 AD-58886 GAAAGAGUGUCUCAUCUUCUU AAGAAGAUGAGACACUCUUUCUG 877-899 3598 3599 AD-58887 AUUUCUGGAACUAGUAAAUUC GAAUUUACUAGUUCCAGAAAUAU 1186-1208 3600 3601 AD-58888 UGUGAUGUGGCCCAUGAGUUU AAACUCAUGGGCCACAUCACACA 1531-1553 3602 3603 AD-58889 AAGAGAGAAGUCCUAUUUCUC GAGAAAUAGGACUUCUCUCUUUC 2208-2230 3604 3605 AD-58890 UGGCAGCACAGAUGAAUCAGA UCUGAUUCAUCUGUGCUGCCAGG 671-693 3606 3607 AD-58891 AUGAUCGUUUCUUUGAGAAAA UUUUCUCAAAGAAACGAUCAUAC 935-957 3608 3609 AD-58892 UCUGGAACUAGUAAAUUCCAU AUGGAAUUUACUAGUUCCAGAAA 1189-1211 3610 3611 AD-59095 GCCCAUUCUUAUCCCGAGU ACUCGGGAUAAGAAUGGGC 360-382 3612 3613 AD-59096 GGAACCAUGCCUCCAUGAU AUCAUGGAGGCAUGGUUCC 1347-1369 3614 3615 AD-59097 UGGAGUCUGUGCGGAUCCU AGGAUCCGCACAGACUCCA 1794-1816 3616 3617 AD-59098 CACCCACGGGUGUGUGGGA UCCCACACACCCGUGGGUG 1112-1134 3618 3619 AD-59099 GGAGUCUGUGCGGAUCCUA UAGGAUCCGCACAGACUCC 1795-1817 3620 3621 AD-59100 CAAAACUGCCCCAAGAUGA UCAUCUUGGGGCAGUUUUG 428-450 3622 3623 AD-59101 GCCUCCAUGAUCCAAGGGA UCCCUUGGAUCAUGGAGGC 1355-1377 3624 3625 AD-59102 CAUCAUCCCUGUGCGGGUU AACCCGCACAGGGAUGAUG 1921-1943 3626 3627 AD-59103 ACCCACGGGUGUGUGGGGA UCCCCACACACCCGUGGGU 1113-1135 3628 3629 AD-59104 CACAUCAUCCCUGUGCGGA UCCGCACAGGGAUGAUGUG 1919-1941 3630 3631 AD-59105 CAGAAAGAGUGUCUCAUCU AGAUGAGACACUCUUUCUG 873-895 3632 3633 AD-59106 CCUCCAUGAUCCAAGGGAU AUCCCUUGGAUCAUGGAGG 1356-1378 3634 3635 AD-59107 UGCCCAUUCUUAUCCCGAA UUCGGGAUAAGAAUGGGCA 359-381 3636 3637 AD-59108 CUUCACCCUGGCUAAGAUA UAUCUUAGCCAGGGUGAAG 1297-1319 3638 3639 AD-59109 AUCAUCCCUGUGCGGGUUA UAACCCGCACAGGGAUGAU 1922-1944 3640 3641 AD-59110 AGAAAGAGUGUCUCAUCUU AAGAUGAGACACUCUUUCU 874-896 3642 3643 AD-59111 CUCCAUGAUCCAAGGGAUU AAUCCCUUGGAUCAUGGAG 1357-1379 3644 3645 AD-59112 CCAUUCUUAUCCCGAGUCA UGACUCGGGAUAAGAAUGG 362-384 3646 3647 AD-59113 CACCCUGGCUAAGAUGAUA UAUCAUCUUAGCCAGGGUG 1300-1322 3648 3649 AD-59114 UCAUCCCUGUGCGGGUUGA UCAACCCGCACAGGGAUGA 1923-1945 3650 3651 AD-59115 AAGAGUGUCUCAUCUUCUU AAGAAGAUGAGACACUCUU 877-899 3652 3653 AD-59116 GUCAUGCCAAAAAUGGACA UGUCCAUUUUUGGCAUGAC 1631-1653 3654 3655 AD-59117 CAUUCUUAUCCCGAGUCCA UGGACUCGGGAUAAGAAUG 363-385 3656 3657 AD-59118 ACCCUGGCUAAGAUGAUGA UCAUCAUCUUAGCCAGGGU 1301-1323 3658 3659 AD-59119 CUCUUCACCCUGGCUAAGA UCUUAGCCAGGGUGAAGAG 1295-1317 3660 3661 AD-59120 AUGCCAAAAAUGGACAUCA UGAUGUCCAUUUUUGGCAU 1634-1656 3662 3663 AD-59121 UGCCCCAAGAUGAUGGAAU AUUCCAUCAUCUUGGGGCA 434-456 3664 3665 AD-59122 GAACCAUGCCUCCAUGAUA UAUCAUGGAGGCAUGGUUC 1348-1370 3666 3667 AD-59123 UCUUCACCCUGGCUAAGAU AUCUUAGCCAGGGUGAAGA 1296-1318 3668 3669 AD-59124 UGCCAAAAAUGGACAUCAU AUGAUGUCCAUUUUUGGCA 1635-1657 3670 3671 AD-59125 CCAGAAAGAGUGUCUCAUA UAUGAGACACUCUUUCUGG 872-894 3672 3673 AD-59126 GAAACUGUCCAUUCAAUGA UCAUUGAAUGGACAGUUUC 1481-1503 3674 3675 AD-59127 UCACCCUGGCUAAGAUGAU AUCAUCUUAGCCAGGGUGA 1299-1321 3676 3677 AD-59128 CCCUGGAGUCUGUGCGGAU AUCCGCACAGACUCCAGGG 1791-1813 3678 3679 AD-59129 GAAAGAGUGUCUCAUCUUA UAAGAUGAGACACUCUUUC 875-897 3680 3681 AD-59130 UGGAGCCCUGGAGUCUGUA UACAGACUCCAGGGCUCCA 1786-1808

The results of the in vitro assays are provided in Table 16. Table 16 also notes the target species of each of the siRNAs.

TABLE 16 Results of Functional Assays Cyno Free Uptake Cyno Transfection Hep3b Transfection Target 1 uM 20 nM 20 nM 0.2 nM 10 nM 0.1 nM Duplex ID Species Type Avg 500 nM Avg 10 nM Avg Avg Avg Avg AD-58848 M/R/Rh/H 21/23 131.6 176.0 104.4 128.0 43.5 44.8 25.3 76.8 AD-58849 H/Rh 21/23 91.9 88.1 92.2 105.0 29.4 35.4 11.5 47.1 AD-58850 H/Rh 21/23 79.4 103.4 80.0 111.2 NA 62.2 31.3 72.0 AD-58851 H/Rh 21/23 99.7 74.7 94.8 104.7 NA 40.7 8.6 81.3 AD-58852 H/Rh 21/23 108.1 91.8 103.3 111.9 101.1 128.8 43.4 129.0 AD-58853 H/Rh 21/23 74.8 67.7 84.2 93.5 24.7 52.9 14.1 61.2 AD-58854 H/Rh 21/23 145.9 124.1 106.6 115.3 119.0 83.9 85.0 84.0 AD-58855 H/Rh 21/23 81.5 97.9 92.7 101.8 39.5 40.3 15.3 67.6 AD-58856 H/Rh 21/23 74.1 90.6 84.6 82.6 22.4 30.7 8.7 33.3 AD-58857 H/Rh 21/23 64.7 91.4 62.3 87.1 22.0 31.6 9.8 106.3 AD-58858 H/Rh 21/23 67.4 91.7 68.6 98.3 27.9 40.3 17.4 44.8 AD-58859 H/Rh 21/23 71.2 77.2 92.4 90.1 19.1 34.3 13.1 39.7 AD-58861 H/Rh 21/23 104.6 107.2 102.0 100.6 25.9 35.1 18.0 69.8 AD-58862 H/Rh 21/23 66.8 77.0 68.7 88.5 20.3 31.1 24.2 49.9 AD-58863 H/Rh 21/23 70.8 66.8 76.8 98.5 21.5 29.7 8.7 54.9 AD-58864 H/Rh 21/23 76.2 85.6 83.7 100.8 60.4 61.0 56.4 87.3 AD-58865 H/Rh 21/23 67.9 77.9 95.9 98.4 21.3 38.6 15.5 81.4 AD-58867 H/Rh 21/23 95.9 93.3 107.0 97.5 32.3 42.7 16.6 79.8 AD-58868 H/Rh 21/23 95.2 92.1 116.2 94.7 54.6 69.2 61.5 105.9 AD-58869 H/Rh 21/23 65.0 78.2 75.8 88.2 17.4 25.0 13.0 63.9 AD-58870 H/Rh 21/23 69.4 92.3 81.0 88.1 29.2 43.8 33.7 79.1 AD-58871 H/Rh 21/23 61.2 77.3 88.2 77.0 71.2 73.2 36.7 110.3 AD-58873 H/Rh 21/23 95.2 100.9 83.3 94.6 54.2 52.8 36.6 73.3 AD-58874 H/Rh 21/23 75.8 76.8 63.8 85.3 22.3 31.2 15.0 38.2 AD-58875 H/Rh 21/23 80.7 88.7 78.6 97.9 48.6 73.6 61.2 90.6 AD-58876 H/Rh 21/23 90.8 93.1 82.5 100.2 41.1 56.9 21.2 58.7 AD-58877 H/Rh 21/23 68.3 85.1 51.2 78.7 18.5 46.6 11.9 27.4 AD-58878 H/Rh 21/23 78.3 68.3 81.2 91.2 24.1 23.4 6.2 37.1 AD-58879 H/Rh 21/23 87.9 94.1 79.7 95.4 32.0 47.8 15.7 82.5 AD-58880 H/Rh 21/23 74.9 72.2 88.9 88.1 20.1 27.5 14.0 60.7 AD-58881 H/Rh 21/23 85.9 76.8 78.8 118.0 22.2 36.7 27.6 71.6 AD-58882 H/Rh 21/23 54.1 53.4 60.3 85.8 14.6 27.2 8.2 23.8 AD-58883 H/Rh 21/23 80.4 69.9 75.7 80.3 31.8 25.8 12.3 63.0 AD-58884 H/Rh 21/23 57.7 55.3 64.8 78.2 20.0 30.0 11.8 68.9 AD-58885 H/Rh 21/23 101.8 91.8 104.1 101.5 85.9 71.9 61.8 71.2 AD-58886 M/R/Rh/H 21/23 47.1 58.0 36.3 93.3 16.0 26.6 9.2 32.0 AD-58887 H/Rh 21/23 73.6 98.7 82.6 95.2 28.5 33.5 12.8 65.2 AD-58888 H/Rh 21/23 90.2 69.9 69.4 85.6 46.9 45.0 16.6 72.0 AD-58889 H/Rh 21/23 83.6 98.6 82.4 92.2 36.5 40.3 31.6 99.4 AD-58890 H/Rh 21/23 69.5 95.4 84.2 88.2 50.8 45.6 21.7 92.9 AD-58891 H/Rh 21/23 62.8 75.7 75.4 109.2 23.6 34.3 15.6 55.8 AD-58892 H/Rh 21/23 60.2 92.9 89.8 92.9 22.8 43.3 20.2 75.6 AD-59095 M/R/Rh/H 19mer 88.9 NA 132.8 NA 48.3 97.4 54.3 99.0 AD-59096 M/R/Rh/H 19mer 95.5 NA 90.5 NA 105.7 138.6 131.4 120.7 AD-59097 M/R/Rh/H 19mer 92.5 NA 84.2 NA 75.0 NA 94.7 108.5 AD-59098 M/R/Rh/H 19mer 84.0 NA 87.7 NA 109.3 NA 130.0 87.3 AD-59099 M/R/Rh/H 19mer 89.7 NA 90.0 NA 77.8 85.4 46.8 74.9 AD-59100 M/R/Rh/H 19mer 84.8 NA 144.3 NA 70.6 108.1 91.5 117.6 AD-59101 M/R/Rh/H 19mer 79.0 NA 103.8 NA 89.8 102.9 124.2 107.0 AD-59102 M/R/Rh/H 19mer 85.9 NA 100.6 NA 72.2 68.5 87.9 95.1 AD-59103 M/R/Rh/H 19mer 86.0 NA 91.1 NA 93.0 81.3 130.0 96.0 AD-59104 M/R/Rh/H 19mer 92.6 NA 96.9 NA 94.9 91.4 124.4 83.1 AD-59105 M/R/Rh/H 19mer 48.9 NA 101.7 NA 18.4 48.9 17.0 34.7 AD-59106 M/R/Rh/H 19mer 63.2 NA 76.7 NA 28.5 40.7 28.6 46.4 AD-59107 M/R/Rh/H 19mer 71.4 NA 68.7 NA 37.1 45.3 26.8 63.6 AD-59108 M/R/Rh/H 19mer 70.7 NA 85.1 NA 89.9 84.8 139.2 101.7 AD-59109 M/R/Rh/H 19mer 86.1 NA 83.4 NA 84.9 96.2 131.7 86.7 AD-59110 M/R/Rh/H 19mer 70.8 NA 119.7 NA 38.5 60.4 67.4 80.3 AD-59111 M/R/Rh/H 19mer 66.1 NA 76.5 NA 52.2 61.0 69.7 87.6 AD-59112 M/R/Rh/H 19mer 71.2 NA 80.2 NA 91.2 83.4 127.4 89.0 AD-59113 M/R/Rh/H 19mer 67.0 NA 77.8 NA 49.1 59.0 66.8 91.4 AD-59114 M/R/Rh/H 19mer 81.7 NA 79.3 NA 96.3 88.0 129.6 72.4 AD-59115 M/R/Rh/H 19mer 40.4 NA 69.6 NA 19.6 35.7 9.3 16.9 AD-59116 M/R/Rh/H 19mer 72.2 NA 78.3 NA 53.5 77.8 70.1 107.8 AD-59117 M/R/Rh/H 19mer 70.7 NA 75.6 NA 75.8 74.9 129.0 103.5 AD-59118 M/R/Rh/H 19mer 68.8 NA 75.9 NA 81.4 82.1 114.1 89.7 AD-59119 M/R/Rh/H 19mer 64.9 NA 86.5 NA 85.1 125.1 122.8 124.8 AD-59120 M/R/Rh/H 19mer 63.5 NA 75.1 NA 29.9 52.0 16.1 54.1 AD-59121 M/R/Rh/H 19mer 67.6 NA 72.0 NA 88.8 77.4 108.0 103.1 AD-59122 M/R/Rh/H 19mer 60.2 NA 62.3 NA 25.1 45.3 16.2 54.8 AD-59123 M/R/Rh/H 19mer 68.6 NA 108.2 NA 59.2 84.6 80.0 97.7 AD-59124 M/R/Rh/H 19mer 47.5 NA 56.5 NA 23.9 40.0 9.8 18.9 AD-59125 M/R/Rh/H 19mer 45.4 NA 47.2 NA 15.2 40.7 14.7 15.1 AD-59126 M/R/Rh/H 19mer 64.3 NA 74.6 NA 51.6 57.1 35.5 54.4 AD-59127 M/R/Rh/H 19mer 103.4 NA 105.8 NA 94.0 156.4 135.9 113.7 AD-59128 M/R/Rh/H 19mer 102.4 NA 81.4 NA 66.3 89.3 60.2 74.9 AD-59129 M/R/Rh/H 19mer 41.3 NA 38.8 NA 17.9 41.4 8.6 12.6 AD-59130 M/R/Rh/H 19mer 58.3 NA 80.8 NA 94.9 78.3 106.7 88.0

Table 17 illustrates the IC₅₀s of select ALAS1 siRNA duplexes. The IC50 were determined from the knockdown of endogenously expressed ALAS1 in the Hep3B cell line, at 24 hours following transfection of each ALAS1 modified siRNA duplex (see Table 14). At least seven duplexes, including AD-58882, AD-58878, AD-58886, AD-58877, AD-59115, AD-58856, and AD-59129, consistently demonstrated IC₅₀s of less than 0.1 nm, indicating that these duplexes were particularly effective in suppressing ALAS1 expression.

TABLE 17 IC_(50S) of select ALAS1 siRNA duplexes Duplex ID 384w IC50 (nM) 96w IC50 (nM) AD-58882 0.008 0.014 AD-58878 0.040 0.031 AD-58886 0.037 0.033 AD-58877 0.031 0.034 AD-59115 0.093 0.052 AD-58856 0.061 0.066 AD-59129 0.085 0.071 AD-59124 0.572 0.078 AD-58874 0.140 0.102 AD-59125 0.118 0.115 AD-59105 0.511 0.144 AD-59120 180.592 0.498 AD-59122 36.646 0.646 AD-59106 7.906 0.847 AD-59126 n/a 1.014 AD-59107 n/a 1.971

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. 

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ALAS1, wherein said dsRNA comprises a sense strand and an antisense strand 15-30 base pairs in length and the antisense strand is complementary to at least 15 contiguous nucleotides of SEQ ID NO: 1 or
 382. 2. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ALAS1, wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to an ALAS1 RNA transcript, which antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the antisense sequence of AD-58882 (SEQ ID NO: 3434) or one of the antisense sequences listed in any one of Tables 2, 3, 6, 7, 8, 9, 14, 15, 18, or
 20. 3. The dsRNA of claim 1, wherein said dsRNA comprises at least one modified nucleotide. 4.-5. (canceled)
 6. The dsRNA of claim 1, wherein the region of complementarity is at least 17 nucleotides in length.
 7. The dsRNA of claim 6, wherein the region of complementarity is between 19 and 23 nucleotides in length.
 8. (canceled)
 9. The dsRNA of claim 1, wherein each strand is no more than 30 nucleotides in length. 10.-11. (canceled)
 12. The dsRNA of claim 1, further comprising a ligand.
 13. The dsRNA of claim 12, wherein said ligand is a GalNAc ligand.
 14. (canceled)
 15. The dsRNA of claim 12, wherein the ligand is conjugated to the 3′ end of the sense strand of the dsRNA.
 16. (canceled)
 17. The dsRNA of claim 1, wherein the dsRNA comprises a sense strand consisting of a sense sequence selected from the sense sequences disclosed in Tables 2, 3, 6, 7, 8, 9, 14, 15, 18 and 20, and an antisense strand consisting of an antisense sequence selected from the antisense sequences disclosed in Tables 2, 3, 6, 7, 8, 9, 14, 15, 18 and
 20. 18. A cell containing the dsRNA of claim
 1. 19. A pharmaceutical composition for inhibiting expression of an ALAS1 gene, the composition comprising the dsRNA of claim
 1. 20.-28. (canceled)
 29. The pharmaceutical composition of claim 19 wherein said composition is administered intravenously or subcutaneously. 30.-31. (canceled)
 32. A method of inhibiting ALAS1 expression in a cell, the method comprising: (a) introducing into the cell the dsRNA of claim 1, and (b) maintaining the cell of step (a) for a time sufficient to obtain degradation of the mRNA transcript of an ALAS1 gene, thereby inhibiting expression of the ALAS1 gene in the cell. 33.-41. (canceled)
 42. A method of treating a disorder related to ALAS1 expression comprising administering to a subject in need of such treatment a therapeutically effective amount of the dsRNA of claim.
 43. (canceled)
 44. The method of claim 42, wherein the subject is at risk for developing, or is diagnosed with, a porphyria. 45.-58. (canceled)
 59. A method for decreasing a level of a porphyrin or a porphyrin precursor in a cell, comprising contacting the cell with the dsRNA of claim 1, in an amount effective to decrease the level of the porphyrin or the porphyrin precursor in the cell. 60.-61. (canceled)
 62. A vector encoding at least one strand of a dsRNA of claim
 1. 63.-65. (canceled)
 66. A cell comprising the vector of claim
 1. 67.-68. (canceled)
 69. The dsRNA of claim 1, wherein the dsRNA comprises a sense strand comprising a sequence selected from the group consisting of SEQ ID NO: 202, SEQ ID NO: 451, SEQ ID NO:330, SEQ ID NO:334, SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, SEQ ID NO:356, SEQ ID NO:358, SEQ ID NO:362, SEQ ID NO:366, SEQ ID NO:376, and SEQ ID NO:380.
 70. The dsRNA of claim 1, wherein the dsRNA comprises an antisense strand comprising a sequence selected from the group consisting of SEQ ID NO: 203, SEO ID NO: 452, SEQ ID NO:331, SEQ ID NO:335, SEQ ID NO:343, SEQ ID NO:345, SEQ ID NO:347, SEQ ID NO:357, SEQ ID NO:359, SEQ ID NO:363, SEQ ID NO:367, SEQ ID NO:377, and SEQ ID NO:381.
 71. The dsRNA of claim 1, wherein the dsRNA comprises a sense strand comprising a sequence selected from the group consisting of SEQ ID NO:140, SEQ ID NO:144, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:172, SEQ ID NO:176, SEQ ID NO:186, and SEQ ID NO:190.
 72. The dsRNA of claim 1, wherein the dsRNA comprises an antisense strand comprising a sequence selected from the group consisting of SEQ ID NO:141, SEQ ID NO:145, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:173, SEQ ID NO:177, SEQ ID NO:187, and SEQ ID NO:191.
 73. The method of claim 42, wherein said method (i) ameliorates a symptom associated with an ALAS1 related disorder (e.g., a porphyria) (ii) inhibits ALAS1 expression in the subject, (iii) decreases a level of a porphyrin precursor or a porphyrin in the subject, (iv) decreases frequency of acute attacks of symptoms associated with a porphyria in the subject, or (v) decreases incidence of acute attacks of symptoms associated with a porphyria in the subject when the subject is exposed to a precipitating factor. 74.-77. (canceled)
 78. A double stranded RNAi (dsRNA) comprising a sense strand complementary to an antisense strand, wherein said antisense strand comprises a region of complementarity to an ALAS1 RNA transcript, wherein each strand has about 14 to about 30 nucleotides, wherein said dsRNA is represented by formula (III): sense: 5′ n_(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n_(q) 3′ (III) antisense: 3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)- N_(a)′-n_(q)′ 5′

wherein: i, j, k, and l are each independently 0 or 1; p, p′, q, and q′ are each independently 0-6; each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof; each n_(p), n_(p)′, n_(q), and n_(q)′ independently represents an overhang nucleotide; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides; modifications on N_(b) differ from the modification on Y and modifications on N_(b)′ differ from the modification on Y′, wherein said dsRNA comprises the sense and antisense sequences of a duplex disclosed in Table 3, 8, 9, or
 15. 79. The dsRNA of claim 78, wherein the sense strand is conjugated to at least one ligand. 80.-88. (canceled)
 89. The dsRNA of claim 78, wherein the modifications on the nucleotides are selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl, and combinations thereof.
 90. The dsRNA of claim 89, wherein the modifications on the nucleotides are 2′-O-methyl, 2′-fluoro or both.
 91. (canceled)
 92. The dsRNA of claim 12, wherein the ligand is attached via a linker.
 93. (canceled)
 94. The dsRNA of claim 12, wherein the ligand is


95. The dsRNA of claim 12, wherein the ligand is attached via a linker, and wherein the ligand and linker are as shown in Formula XXIV:

96.-111. (canceled)
 112. The method of claim 42, wherein the porphyria is a hepatic porphyria selected from acute intermittent porphyria (AIP) hereditary coproporphyria (HCP), variegate porphyria (VP), ALA deyhdratase deficiency porphyria (ADP), and hepatoerythropoietic porphyria. 113.-115. (canceled)
 116. The method of claim 42, wherein the dsRNA or composition comprising dsRNA is administered before, during, or after an acute attack of porphyria.
 117. (canceled)
 118. The method of claim 42, wherein the dsRNA or composition comprising dsRNA is administered during a prodrome.
 119. The method of claim 118, wherein the prodrome is characterized by pain, nausea, psychological symptoms, restlessness or insomnia. 120.-123. (canceled)
 124. The method of claim 42, wherein the subject has an elevated level of ALA and/or PBG. 125.-150. (canceled)
 151. A method of treating a subject with an elevated level of ALA and/or PBG, the method comprising administering to the subject a double-stranded ribonucleic acid (dsRNA), wherein said dsRNA comprises a sense strand and an antisense strand 15-30 base pairs in length and the antisense strand is complementary to at least 15 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:382. 152.-154. (canceled) 